A high-carbon rooting method for Macapuno coconut tissue culture seedlings

Abstract

The present application provides a high-carbon rooting method for Macapuno coconut tissue culture seedlings, belonging to the technical field of coconut tissue culture. The high-carbon rooting method for Macapuno coconut tissue culture seedlings of the present application comprises the following steps: S1, disinfecting the embryo and tools; S2, disinfecting the endosperm block and the embryo; S3, primary dark culture; S4, light culture of the seedlings; S5, acclimatization treatment; S6, preparation before transplantation; S7, transplantation. By using an embryo extractor to extract the embryo, the present application ensures the success rate of embryo extraction of Macapuno coconut; based on the characteristics of Macapuno coconut, a culture medium suitable for Macapuno coconut is specially prepared to ensure the germination rate of zygotic embryo culture; high-sugar culture is adopted during embryo culture, and at the same time, exogenous CO2 and light of different light qualities are added to the culture environment, which improves the growth characteristics of embryo culture seedlings and the survival rate of seedlings, and is beneficial to the popularization and planting of Macapuno coconut.

Description

Technical Field

[0001] This application belongs to the technical field of coconut tissue culture, and relates to a high-carbon rooting method for Macapuno coconut tissue culture seedlings. Background Art

[0002] Macapuno coconut (macapuno, also spelled as makapuno) is a naturally occurring coconut variety with abnormal endosperm development. As a result of this abnormal development, a soft, translucent jelly-like pulp (endosperm) almost fills the entire central cavity of the coconut seed, with little coconut water. Because the endosperm (coconut meat) has a soft texture and tastes soft, glutinous, and sweet, like glutinous rice, it is also called glutinous rice coconut.

[0003] The embryos of Macapuno seeds are normal, but the surrounding abnormal endosperm cannot support their germination, making Macapuno seeds effectively sterile. Therefore, although Macapuno coconut is regarded as a traditional delicacy, it is still rare and expensive. Macapuno coconut is one of the most valuable among all coconut varieties, with a domestic price of over 200 yuan.

[0004] Although planting palm trees with Macapuno coconuts together or separately can increase the yield of Macapuno coconuts, the chance of the phenotype reappearing in the offspring fruits is very low, only 2% to 21%. Summary of the Invention

[0005] In view of this, this application provides a high-carbon rooting method for Macapuno coconut tissue culture seedlings, which has a low embryo contamination rate and a high germination rate, while improving the survival rate and growth characteristics of the seedlings, is conducive to the popularization and planting of Macapuno coconut, and can effectively overcome the defects existing in the above-mentioned prior art.

[0006] This application provides a high-carbon rooting method for Macapuno coconut tissue culture seedlings, including the following steps:

[0007] S1. Disinfection of embryo and tools: Use an embryo extractor to extract endosperm blocks from the coconut meat of Macapuno coconut, and locate the embryo position based on the large hole at the bottom of the coconut;

[0008] S2. Disinfection treatment of endosperm blocks and embryos: Rinse the endosperm blocks thoroughly, then cut the endosperm blocks in a laminar flow hood, take out the embryos, soak the embryos, then rinse with sterile water, and finally dry them in the laminar flow hood to obtain surface-sterilized zygotic embryos;

[0009] S3. Primary dark culture: Place the surface-sterilized zygotic embryos on a solid medium and cultivate them under dark conditions until the embryos start to germinate;

[0010] S4. Seedling light culture: Transfer the germinated embryo to a medium composed of Y3 liquid medium, sucrose, and activated carbon, and culture it under light until leaves grow on the seedlings;

[0011] S5. Hardening-off treatment: Select strong tissue culture seedlings, place them in sugar-free Y3 liquid culture supplemented with activated carbon, and conduct hardening-off under light with controlled CO2 concentration and light intensity;

[0012] S6. Preparation before transplanting: In a laminar flow hood, completely wash off the residual sugar-containing medium on the tissue culture seedlings with sterile water, then soak them, and then transplant the tissue culture seedlings into disposable plastic cups filled with nutrient soil and vermiculite, and place them in an incubator;

[0013] S7. Transplanting: When the seedlings after hardening-off have strong and healthy leaves with roots, transplant them outdoors.

[0014] Preferably, in step S1, the embryo extractor includes an embryo extraction tube. The embryo extraction tube is 20 cm long, with an outer diameter of 3.5 cm and a wall thickness of 0.3 cm. It is made of steel. The front end is thinned to form a wedge shape for easy embedding into the endosperm. The rear part is equipped with a small stick, and pushing forward can push out a cylindrical endosperm block containing the embryo from the front end.

[0015] Preferably, in step S1, when extracting the embryo of Macapuno coconut, insert it at the corresponding position of the endosperm according to the position of the larger one of the three holes at the bottom of the coconut to obtain an endosperm block containing the embryo.

[0016] Preferably, in step S1, before each extraction of the endosperm block, spray the embryo extractor with 70% ethanol for disinfection.

[0017] Preferably, the specific process of step S2 is as follows: Rinse the endosperm block with 2.4% NaOCl for 10 minutes, then cut the endosperm block in a laminar flow hood, take out the embryo, soak the embryo in 70% ethanol for 1 minute, then put it in 0.8% NaOCl for treatment for 10 minutes, then rinse it with sterile water 5 times, 1 minute each time, and finally dry it in a laminar flow hood.

[0018] Preferably, in step S3, the Y3 basal medium contains:

[0019] Y³ Macronutrients (mg / L): Potassium nitrate KNO₃ 2000.00, Potassium chloride KCl 1500.00, Ammonium chloride NH₄Cl 530.00, Sodium dihydrogen phosphate NaH₂PO₄·2H₂O 310.00, Calcium chloride CaCl₂·2H₂O 295.00, Magnesium sulfate MgSO₄·7H₂O 250.00;

[0020] Y3 Micronutrients (mg / L): Manganese sulfate MnSO4ꞏ4H2O 11.50, Potassium iodide KI 8.00, Zinc sulfate ZnSO4ꞏ7H2O 7.30, Boric acid H3BO3 3.00, Copper sulfate CuSO4ꞏ5H2O 0.270, Cobalt chloride CoCl2ꞏ6H2O 0.250, Sodium molybdate NaMoO4·H2O 0.250, Nickel chloride NiClꞏ6H2O 0.026, Ferrous sulfate Fe2SO4ꞏ7H2O 42.00, Disodium ethylenediaminetetraacetate Na2EDTA 56.0;

[0021] Vitamins and Amino Acids (mg / L): Pyridoxine hydrochloride (Vitamin B6) C8H 11 NO3ꞏHCl 0.05, Thiamine hydrochloride (Vitamin B1) C 12 H 17 ClN4OSꞏHCl 0.05, Niacin (Vitamin B3) C6H5NO2 0.05, Calcium pantothenate (Vitamin B5) (C9H 16 NO5)2Ca 0.05, Biotin (Vitamin H) C 10 H 16 N2O3S 0.05, Folic acid (Vitamin Bc, Vitamin M) C 19 H 19 N7O6 0.05, Glycine C2H5NO2 1.00;

[0022] Other Additives: Abscisic acid (ABA(a)) C 15 H 20 O4 0.25 mg / L, α-Naphthaleneacetic acid (NAA(b)) C 12 H 10 O2 18.6 mg / L, Sucrose C6H 22 O 11 50 - 60.0 g / L, Activated carbon 2.0 g / L.

[0023] Preferably, in step S3, the whole process of tissue culture uses a sucrose content of 60 g / L. The Y3 basal medium also contains 7.0 g / L of agar or 3.0 g / L of Gelzan. The pH values of the Y3 basal medium and the solid medium are 5.7.

[0024] Preferably, the specific process of step S3 is as follows: Place the surface-sterilized zygotic embryos on a solid medium composed of Y3 basal medium, 60 g / L sucrose, 2 g / L activated carbon, and 7 g / L agar, and cultivate them under dark conditions at 27 ± 2 °C until the embryos start to germinate; after germination, subculture 2 - 3 times, and use 60 g / L of high sugar throughout the process without reducing the sugar content; after rooting, dilute with a commercial tissue culture antifungal agent, soak the seedlings for 8 h, wash them once with sugar-free Y3 medium, and then transfer them to the culture substrate. The composition of the culture substrate is commercial nutrient soil + vermiculite, with a composition of 50% nutrient soil and 50% vermiculite.

[0025] Preferably, the specific process of step S4 is as follows: Transfer the germinated embryos to a medium composed of Y3 liquid medium, 60 g / L sucrose, and 2 g / L activated carbon, and use red and blue LED lights in a 7:3 ratio, with a light intensity of 25 μmol m -2 s -1 , illuminate for 16 hours every day, and culture at 27 ± 1 °C for 8 - 12 weeks until the seedlings grow 1 - 2 leaves; or

[0026] The specific process of step S5 is as follows: Select robust tissue culture seedlings with a main root ≥ 5 cm and ≥ 20 fibrous roots, place them in a sugar-free Y3 liquid medium with 2 g / L activated carbon, control the CO2 concentration at 1600 μmol / mol, and use red and blue LED lights with a ratio of 7:3 and a light intensity of 600 μmol m -2 s -1 for illumination, and illuminate for 16 hours every day for seedling hardening.

[0027] Preferably, the specific process of step S6 is as follows: In a laminar flow hood, completely wash the sugar-containing culture medium residue on the tissue culture seedlings with sterile water, and then soak them in a 1 g / L carbendazim dilution, using 60 mL for each seedling for sterilization, and the sterilization time is 8 hours; after sterilization, transplant the tissue culture seedlings into disposable plastic cups filled with nutrient soil and vermiculite, and place them in an incubator; or

[0028] The specific process of step S7 is as follows: When the seedlings after hardening have more than 3 leaves and strong roots, transplant them outdoors.

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] (1) The present application ensures the success rate of embryo extraction from Macapuno coconut by using an embryo extractor; based on the characteristics of Macapuno coconut, a culture medium suitable for Macapuno coconut is specially formulated to ensure the germination rate of zygotic embryo culture; high sugar culture is used in the embryo culture process, and at the same time, exogenous CO2 and light of different light qualities are added to the culture environment, which improves the growth characteristics and survival rate of embryo culture seedlings, and is conducive to the promotion and planting of Macapuno coconut.

[0031] (2) This method shortens the time required for the application, reducing the time for secondary roots to fully grow to 4-5 months, increasing the survival rate of seedlings after hardening to 94%, and reducing the fungal infection rate to 17%. Compared with traditional methods, the fresh weight of seedlings, stem diameter, and leaf width increased by 51.8%, 74%, and 44.9%, respectively. The technical effect is significant and has the potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the present application or the prior art, the following briefly introduces the drawings required for use in the description of the present application or the prior art. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0033] Figure 1 A photo of embryo-cultured seedlings using continuous high-sugar tissue culture (left) four months after embryo culture; compared to a photo of tissue-cultured seedlings using reduced-sugar tissue culture (right); the difference in the upper root portion between the two is not significant, but the root development is quite different.

[0034] Figure 2 For the embryo culture of seedlings using the method described in prior art 202410635327.X, Figure 2 (A) is a photo of seedlings that have been hardened; Figure 2 (B) A photo of the sterilized seedlings being transplanted into the prepared tissue culture medium; Figure 2 (C) A photo of a seedling placed in a substrate with plastic sheeting covering the top to reduce water loss.

[0035] Figure 3 Photos of tissue culture seedlings that have successfully taken root in vitro (aseptically) using the method of this application can be transferred to Figure 2 In the matrix shown. DETAILED DESCRIPTION

[0036] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in this application will be clearly and completely described below in conjunction with the accompanying drawings in this application. Apparently, the described embodiments are some, but not all, of the embodiments of this application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in this application without creative efforts fall within the scope of protection of this application.

[0037] Unless otherwise specified, the experimental methods used in the embodiments of this application are all conventional methods.

[0038] In the following embodiments and comparative examples, unless otherwise specified, all raw materials can be obtained through commercial purchase or conventional methods.

[0039] 1. Material Preparation

[0040] ● Plant material: Mature Marapuama coconut fruits (endosperm is semi-transparent and gelatinous).

[0041] ● Sterilization reagents: 2.4% NaClO solution, 70% ethanol, 0.8% NaClO solution, 1 g / L carbendazim diluent (commercial).

[0042] ● Culture medium: Based on the Y3 culture medium formula (Table 1), the sucrose concentration is fixed at 60 g / L throughout the process, containing 2 g / L activated carbon, 18.6 mg / L NAA, and pH 5.7.

[0043] Table 1 Composition of high-carbon culture medium

[0044] Example 1

[0045] The high-carbon rooting method for Marapuama coconut tissue culture seedlings in this example includes the following steps:

[0046] S1. Disinfection of embryo and tools: Use an embryo extractor to extract endosperm blocks from the coconut meat of Marapuama coconut, and locate the embryo position according to the large hole at the bottom of the coconut;

[0047] Among them, the embryo extractor includes an embryo extraction tube. The embryo extraction tube is 20 cm long, with an outer diameter of 3.5 cm and a wall thickness of 0.3 cm. It is made of steel. The front end is thinned to form a wedge shape for easy insertion into the endosperm. The rear part is equipped with a small stick, and pushing forward can push out the cylindrical endosperm block containing the embryo from the front end;

[0048] Among them, when taking the embryo of Marapuama coconut, insert it at the corresponding position of the endosperm according to the position of the larger one of the three holes at the bottom of the coconut to obtain the endosperm block containing the embryo;

[0049] Among them, before each extraction of the endosperm mass, the embryo extractor is sprayed with 70% ethanol for disinfection.

[0050] S2. Disinfection treatment of the endosperm mass and the embryo: The endosperm mass is rinsed with 2.4% NaOCl for 10 minutes, then the endosperm mass is cut open in a laminar flow hood to take out the embryo. The embryo is soaked in 70% ethanol for 1 minute, then placed in 0.8% NaOCl for 10 minutes, and then rinsed with sterile water 5 times, 1 minute each time. Finally, it is dried in a laminar flow hood to obtain surface-sterilized zygotic embryos.

[0051] S3. Primary dark culture: The surface-sterilized zygotic embryos are placed on a solid medium composed of Y3 basal medium, 60 g / L sucrose, 2 g / L activated carbon, and 7 g / L agar, and cultured under dark conditions at 27 ± 2 °C until the embryos start to germinate; after germination, subculture is carried out 2 - 3 times. Throughout the process, 60 g / L of high sugar is used without sugar reduction treatment; after rooting, the seedlings are diluted with a commercial tissue culture antifungal agent and soaked for 8 h, then washed once with sugar-free Y3 medium and transferred to the culture substrate. The composition of the culture substrate is commercial nutrient soil + vermiculite, with a composition of 50% nutrient soil and 50% vermiculite;

[0052] Among them, the Y3 basal medium contains: Y3 macronutrients (mg / L): potassium nitrate KNO3 2000.00, potassium chloride KCl 1500.00, ammonium chloride NH4Cl 530.00, sodium dihydrogen phosphate NaH2PO4·2H2O 310.00, calcium chloride CaCl2·2H2O 295.00, magnesium sulfate MgSO4·7H2O 250.00; Y3 micronutrients (mg / L): manganese sulfate MnSO4·4H2O 11.50, potassium iodide KI 8.00, zinc sulfate ZnSO4·7H2O 7.30, boric acid H3BO3 3.00, copper sulfate CuSO4·5H2O 0.270, cobalt chloride CoCl2·6H2O 0.250, sodium molybdate NaMoO4·H2O 0.250, nickel chloride NiCl·6H2O 0.026, ferrous sulfate Fe2SO4·7H2O 42.00, disodium ethylenediaminetetraacetate Na2EDTA 56.0; vitamins and amino acids (mg / L): pyridoxine hydrochloride (vitamin B6) C8H 11 NO3·HCl 0.05, thiamine hydrochloride (vitamin B1) C 12 H 17 ClN4OS·HCl 0.05, nicotinic acid (vitamin B3) C6H5NO2 0.05, calcium pantothenate (vitamin B5) (C9H 16NO5) 2Ca 0.05, biotin (vitamin H) C 10 H 16 N2O3S 0.05, folic acid (vitamin Bc, vitamin M) C 19 H 19 N7O6 0.05, glycine C2H5NO2 1.00; Other additives: abscisic acid (ABA(a)) C 15 H 20 O4 0.25 mg / L, α-naphthaleneacetic acid (NAA(b)) C 12 H 10 O2 18.6mg / L, sucrose C6H 22 O 11 50~60.0 g / L, activated carbon 2.0 g / L;

[0053] Among them, sucrose content of 60 g / L is used throughout the tissue culture process. The Y3 basal medium also contains 7.0 g / L of agar or 3.0 g / L of Gelzan. The pH values of the Y3 basal medium and the solid medium are 5.7.

[0054] S4. Seedling light culture: Transfer the germinated embryo to a medium composed of Y3 liquid medium, 60 g / L of sucrose and 2 g / L of activated carbon, and irradiate it with red and blue LEDs at a ratio of 7:3. The light intensity is 25 μmol m -2 s -1 , with 16 hours of light per day, and culture it for 8 - 12 weeks at 27±1°C until the seedlings grow 1 - 2 leaves.

[0055] S5. Hardening-off treatment: Select healthy tissue culture seedlings with a main root ≥5 cm and ≥20 fibrous roots, place them in a Y3 liquid medium without sugar and with 2 g / L of activated carbon, control the CO2 concentration at 1600 μmol / mol, and use red and blue LEDs with a ratio of 7:3 and a light intensity of 600 μmol m -2 s -1 LED light for 16 hours of light per day for hardening-off.

[0056] S6. Preparation before transplantation: In a laminar flow hood, completely wash the sugar-containing medium residue on the tissue culture seedlings with sterile water, then soak them in a 1 g / L carbendazim dilution, and use 60 mL for each seedling for sterilization for 8 hours; After sterilization, transplant the tissue culture seedlings into disposable plastic cups filled with nutrient soil and vermiculite, and place them in an incubator.

[0057] S7. Transplantation: When the seedlings after hardening-off have more than 3 leaves and strong roots, transplant them outdoors.

[0058] In this example, through full-course high-sugar culture (60 g / L sucrose), enhanced sterilization (soaking with carbendazim), and light-CO2 synergistic regulation, the rooting period of in vitro seedlings of Macapuno coconut was significantly shortened to 4 - 5 months, the survival rate of acclimatized seedlings was increased to 94%, and the fungal infection rate was reduced to 17%. Compared with the traditional method, the fresh weight, stem diameter, and leaf width of the seedlings were increased by 51.8%, 74%, and 44.9% respectively, with significant technical effects and potential for industrial application.

[0059] Comparative Example 1

[0060] The technical solution of this comparative example refers to the prior art 202410635327.X.

[0061] Test Example

[0062] A total of 60 embryos were used in the experiment (30 for each method), and the method of this application was compared with the method in Comparative Example 1. After the comparative experiment, the relevant comparative data are shown in Tables 2 - 4.

[0063] Table 2

[0064]

[0065] Table 3

[0066]

[0067] Table 4

[0068]

[0069] Compared with Comparative Example 1, the method of this application has the following advantages:

[0070] 1. Natural nutrient supply system: The nutrient soil is rich in humus, microbial communities, and basic nutrients such as nitrogen, phosphorus, and potassium. Vermiculite has the ability of ion exchange, which can slowly release nutrients and reduce the frequency of topdressing in the initial stage.

[0071] 2. Dynamic buffering and water and fertilizer retention: The organic matter structure of the nutrient soil can regulate pH fluctuations (natural pH 5.8 - 6.8). Vermiculite adsorbs water and nutrients through layered pores, and the water holding rate reaches 40 - 50%, significantly reducing the risk of drought stress.

[0072] 3. Disease resistance and root development optimization: The decomposed nutrient soil contains natural antagonistic microbial communities. The porous structure of vermiculite inhibits the reproduction of pathogenic bacteria and provides a loose growth environment for the roots at the same time.

[0073] The nutrient soil is rich in nitrogen, phosphorus, and potassium nutrients, and vermiculite has the ability of ion exchange. The combination of the two can continuously supply fertilizers for tissue culture seedlings and reduce the frequency of topdressing. The nutrient soil can adjust the pH, and vermiculite can adsorb water and nutrients, reducing the risk of drought stress for seedlings. Its water holding rate reaches 40 - 50%. Moreover, this combination can inhibit the reproduction of pathogenic bacteria and facilitate root development. Although its cost is 7.5 - 13 yuan per kilogram, which is higher than that of rice husk charcoal + coconut coir substrate, it can increase the root biomass by 10 - 15%.

[0074] At the same time, we found that the combination of rice husk charcoal + coconut coir are both organic matters. When contaminated, they are extremely likely to spread, and in severe cases, it even endangers the growth of seedlings. When tissue culture seedlings are directly placed into the rice husk charcoal + coconut coir substrate full of microorganisms and pathogens without being soaked in disinfectant, the risk is extremely high. While the nutrient soil and vermiculite contain less organic matter, and at the same time, the tissue culture seedlings soaked and sterilized hardly carry pathogenic bacteria, which can greatly improve the survival rate.

[0075] From Figure 1 it can be seen the difference in rooting between using high - sugar culture and the old method at the same growth stage. Figure 2 For embryo - cultured seedlings acclimatized by the method described in the prior art 202410635327.X Figure 2 (A) is a photo of the seedlings that have been acclimatized; Figure 2 (B) is a photo of the disinfected seedlings transferred into the prepared tissue culture substrate; Figure 2 (C) is a photo of the upper part of the seedlings placed in the substrate covered with a plastic sheet to reduce water loss. Figure 3 It is a photo of tissue culture seedlings that have successfully rooted in vitro (sterile) by the method of this application and can be transferred to Figure 2 the substrate shown. From Figure 3 it can be seen that almost all the seedlings using the method of this application have grown a large number of roots, greatly improving the survival rate.

[0076] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of this application, not to limit it; although this application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A high-carbon rooting method for Macapuno coconut tissue culture seedlings, characterized in that, It includes the following steps: S1. Embryo and tool disinfection: Use an embryo extractor to extract the endosperm block from the coconut meat of Macapuno coconut, and locate the embryo position based on the large hole at the bottom of the coconut; S2. Disinfection treatment of the endosperm block and embryo: Rinse the endosperm block thoroughly, then cut the endosperm block in a laminar flow hood, take out the embryo, soak the embryo, then rinse it with sterile water, and finally dry it in the laminar flow hood to obtain a surface-sterilized zygotic embryo; S3. Primary dark culture: Place the surface-sterilized zygotic embryo on a solid medium and cultivate it in the dark until the embryo begins to germinate; S4. Seedling light culture: Transfer the germinated embryo to a medium composed of Y3 liquid medium, sucrose and activated carbon, and culture it under light until the seedling grows leaves; S5. Hardening-off treatment: Select healthy tissue-cultured seedlings, place them in Y3 liquid culture without sugar and with activated carbon, and carry out hardening-off under light with controlled CO2 concentration and light intensity; S6. Preparation before transplantation: In a laminar flow hood, completely wash the residual sugar-containing medium on the tissue-cultured seedlings with sterile water, then soak them, and then transplant the tissue-cultured seedlings into a disposable plastic cup filled with nutrient soil and vermiculite, and place them in an incubator; S7. Transplantation: When the seedlings after hardening-off have strong-rooted leaves, transplant them outdoors; The specific process of step S6 is as follows: In a laminar flow hood, completely wash the residual sugar-containing medium on the tissue-cultured seedlings with sterile water, then soak them in a 1g / L carbendazim dilution, use 60mL for each seedling for sterilization, and the sterilization time is 8 hours; after sterilization, transplant the tissue-cultured seedlings into a disposable plastic cup filled with nutrient soil and vermiculite, and place them in an incubator; The specific process of step S3 is as follows: Place the surface-sterilized zygotic embryo on a solid medium composed of Y3 liquid medium, 60g / L sucrose, 2g / L activated carbon, and 7g / L agar, and cultivate it in the dark at 27 ± 2°C until the embryo begins to germinate; after germination, subculture 2-3 times, use 60g / L high sugar throughout the process without sugar reduction treatment; after rooting, dilute with a commercial tissue-culture antifungal agent, soak the seedlings for 8h, wash them once with sugar-free Y3 medium, and then transfer them to the culture medium. The composition of the culture medium is commercial nutrient soil + vermiculite, with a composition of 50% nutrient soil and 50% vermiculite; The specific process of step S4 is as follows: Transfer the germinated embryo to a culture medium composed of Y3 liquid medium, 60 g / L sucrose, and 2 g / L activated carbon, and irradiate it with red and blue LEDs in a 7:3 ratio. The light intensity is 25 μmol m -2 s -1 , irradiate for 16 hours every day, and culture at 27 ± 1 °C for 8 - 12 weeks until the seedlings grow 1 - 2 leaves; The specific process of step S5 is as follows: select robust tissue culture seedlings with a main root ≥ 5 cm and ≥ 20 fibrous roots, place them in Y3 liquid medium without sugar and with 2 g / L activated carbon, control the CO2 concentration at 1600 μmol / mol, use LED light with a red-blue ratio of 7:3 and a light intensity of 600 μmol m -2 s -1 for 16 hours of lighting every day for seedling hardening.

2. The high-carbon rooting method of the macapuno coconut tissue culture seedlings according to claim 1, wherein In step S1, the embryo extractor includes an embryo extraction tube. The embryo extraction tube is 20 cm long, with an outer diameter of 3.5 cm and a wall thickness of 0.3 cm. It is made of steel. The front end is thinned to form a wedge shape for easy embedding in the endosperm. The rear part is equipped with a small stick, and pushing forward can push out a cylindrical endosperm block containing the embryo from the front end.

3. The high-carbon rooting method of the macapuno coconut tissue culture seedlings according to claim 1, characterized in that, In step S1, when taking the embryo of Macapuno coconut, insert it at the corresponding position of the endosperm according to the position of the larger one of the three holes at the bottom of the coconut to obtain an endosperm block containing the embryo.

4. The high-carbon rooting method of Macapuno coconut tissue culture seedlings according to claim 1, characterized in that In step S1, spray the embryo extractor with 70% ethanol for disinfection before each extraction of the endosperm block.

5. The high-carbon rooting method for Macapuno coconut tissue culture seedlings according to claim 1, characterized in that The specific process of step S2 is as follows: Rinse the endosperm mass with 2.4% NaOCl for 10 minutes, then cut the endosperm mass in a laminar flow hood, take out the embryo, soak the embryo in 70% ethanol for 1 minute, then place it in 0.8% NaOCl for treatment for 10 minutes, then rinse it with sterile water 5 times, 1 minute each time, and finally dry it in the laminar flow hood.

6. The high-carbon rooting method of the Macapuno coconut tissue culture seedlings according to claim 1, characterized in that, In step S3, the Y3 liquid medium contains: Y3 macronutrients: potassium nitrate KNO3 2000.00 mg / L, potassium chloride KCl 1500.00 mg / L, ammonium chloride NH4Cl 530.00 mg / L, sodium dihydrogen phosphate NaH2PO4·2H2O 310.00 mg / L, calcium chloride CaCl2·2H2O 295.00 mg / L, magnesium sulfate MgSO4·7H2O 250.00 mg / L; Y3 micronutrients: manganese sulfate MnSO4·4H2O 11.50 mg / L, potassium iodide KI 8.00 mg / L, zinc sulfate ZnSO4·7H2O 7.30 mg / L, boric acid H3BO3 3.00 mg / L, copper sulfate CuSO4·5H2O 0.270 mg / L, cobalt chloride CoCl2·6H2O 0.250 mg / L, sodium molybdate NaMoO4·H2O 0.250 mg / L, nickel chloride NiCl·6H2O 0.026 mg / L, ferrous sulfate Fe2SO4·7H2O 42.00 mg / L, disodium ethylenediaminetetraacetate Na2EDTA 56.0 mg / L; Vitamins and Amino Acids: Pyridoxine Hydrochloride C8H 11 NO3·HCl 0.05 mg / L, Thiamine Hydrochloride C 12 H 17 ClN4OS·HCl 0.05 mg / L, Nicotinic Acid C6H5NO2 0.05 mg / L, Calcium Pantothenate (C9H 16 NO5)2Ca 0.05 mg / L, Biotin C 10 H 16 N2O3S 0.05 mg / L, Folic Acid C 19 H 19 N7O6 0.05 mg / L, Glycine C2H5NO2 1.00 mg / L; Other additives: abscisic acid (ABA(a)) C 15 H 20 O4 0.25 mg / L, α-naphthaleneacetic acid (NAA(b)) C 12 H 10 O2 18.6mg / L, sucrose C6H 22 O 11 50~60.0 g / L, activated carbon 2.0 g / L.

7. The high-carbon rooting method for Macapuno coconut tissue culture seedlings according to claim 6, characterized in that, In step S3, a sucrose content of 60 g / L is used throughout the tissue culture process. The Y3 liquid medium also contains 7.0 g / L of agar or 3.0 g / L of Gelzan. The pH value of the Y3 liquid medium and the solid medium is 5.

7.

8. The high-carbon rooting method for Macapuno coconut tissue culture seedlings according to claim 1, characterized in that, The specific process of step S7 is as follows: When the seedlings after acclimatization have more than 3 leaves and strong roots, transplant them outdoors.

Images