High-carbon rooting method of macapnol coconut tissue culture seedlings

Abstract

The invention provides a high-carbon rooting method for macapono coconut tissue culture seedlings, and belongs to the technical field of coconut tissue culture. The high-carbon rooting method of macapono coconut tissue culture seedlings comprises the following steps: S1, taking embryos and disinfecting tools; s2, disinfecting endosperm blocks and embryos; s3, primary dark culture; s4, seedling illumination culture; s5, seedling hardening treatment; s6, preparation before transplanting; and S7, transplanting. According to the method, the embryo taking device is used for taking the embryo, so that the embryo taking success rate of macapono coconuts is ensured; on the basis of the characteristics of MACP coconuts, a culture medium suitable for MACP coconuts is specially prepared, and the germination rate of zygotic embryo culture is guaranteed; high-sugar culture is adopted in the embryo culture process, meanwhile, exogenous CO2 and illumination of different light qualities are added in the culture environment, the growth character of embryo culture seedlings is improved, the survival rate of the seedlings is increased, and popularization and planting of Macapuno coconuts are facilitated.

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. The result of this abnormal development is that a soft, translucent jelly-like pulp (endosperm) almost fills the entire central cavity of the coconut seed, with little coconut water. Since 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, facilitating the popularization and planting of macapuno coconut, and effectively overcoming 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: 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; S2. Disinfection treatment of endosperm blocks and embryos: Rinse the endosperm blocks thoroughly, then cut the endosperm blocks in a super clean bench, take out the embryos, soak the embryos, then rinse with sterile water, and finally dry them in the super clean bench to obtain surface-sterilized zygotic embryos; S3. Primary dark culture: Place the surface-sterilized zygotic embryos on a solid medium and cultivate them in the dark until the embryos start to germinate; S4. Seedling light culture: Transfer the germinated embryos to a medium composed of Y3 liquid medium, sucrose, and activated carbon, and culture them under light until the seedlings grow leaves; S5. Acclimatization treatment: Select healthy tissue culture seedlings and place them in a sugar-free Y3 liquid culture supplemented with activated carbon, and carry out acclimatization under controlled CO 2 concentration and light intensity; S6. Preparation before transplantation: In a laminar flow hood, thoroughly wash the sugar-containing culture medium residues 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; S7. Transplantation: When the seedlings after acclimatization have strong and healthy leaves with roots, transplant them outdoors.

[0007] Preferably, in step S1, the embryo extractor includes an embryo extraction tube, the embryo extraction tube is 20 cm long, has an outer diameter of 3.5 cm, a wall thickness of 0.3 cm, is made of steel, and the front end is thinned to form a wedge shape for convenient embedding into the endosperm. The rear part is provided with a small stick, and pushing forward can push out a cylindrical endosperm block containing the embryo from the front end.

[0008] Preferably, in step S1, when extracting the embryo of Macapuno coconut, according to the position of the larger one of the three holes at the bottom of the coconut, insert it at the corresponding position of the endosperm to obtain an endosperm block containing the embryo.

[0009] Preferably, in step S1, before each extraction of the endosperm block, spray the embryo extractor with 70% ethanol for disinfection.

[0010] Preferably, the specific process of step S2 is: 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 place it in 0.8% NaOCl for 10 minutes, then rinse it with sterile water 5 times, 1 minute each time, and finally dry it in a laminar flow hood.

[0011] Preferably, in step S3, the Y3 basal medium contains: Y3 macronutrients (mg / L): potassium nitrate KNO 3 2000.00, potassium chloride KCl 1500.00, ammonium chloride NH 4 Cl 530.00, sodium dihydrogen phosphate NaH 2 PO 4 ꞏ2H 2 O 310.00, calcium chloride CaCl 2 ꞏ2H 2 O 295.00, magnesium sulfate MgSO 4 ꞏ7H 2 O 250.00; Y3 micronutrients (mg / L): manganese sulfate MnSO 4 ꞏ4H2 O 11.50, potassium iodide KI 8.00, zinc sulfate ZnSO 4 ꞏ7H 2 O 7.30, boric acid H 3 BO 3 3.00, copper sulfate CuSO 4 ꞏ5H 2 O 0.270, cobalt chloride CoCl 2 ꞏ6H 2 O 0.250, sodium molybdate NaMoO 4 ·H 2 O 0.250, nickel chloride NiClꞏ6H 2 O 0.026, ferrous sulfate Fe 2 SO 4 ꞏ7H 2 O 42.00, disodium ethylenediaminetetraacetate Na 2 EDTA 56.0; Vitamins and amino acids (mg / L): pyridoxine hydrochloride (vitamin B6) C 8 H 11 NO 3 ꞏHCl 0.05, thiamine hydrochloride (vitamin B1) C 12 H 17 ClN 4 OSꞏHCl 0.05, nicotinic acid (vitamin B3) C 6 H 5 NO 2 0.05, calcium pantothenate (vitamin B5) (C 9 H 16 NO 5 ) 2 Ca 0.05, biotin (vitamin H) C 10 H 16 N 2 O 3 S 0.05, folic acid (vitamin Bc, vitamin M) C 19 H 19 N 7 O 6 0.05, glycine C 2 H 5 NO 2 1.00; Other additives: abscisic acid (ABA(a)) C 15 H 20 O 4 0.25 mg / L, α-naphthaleneacetic acid (NAA(b)) C 12 H 10O 2 18.6 mg / L, sucrose C 6 H 22 O 11 50 - 60.0 g / L, activated carbon 2.0 g / L.

[0012] 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.

[0013] Preferably, the specific process of step S3 is: placing the surface - sterilized zygotic embryo on a solid medium composed of Y3 basal medium, 60 g / L of sucrose, 2 g / L of activated carbon, and 7 g / L of agar, and culturing it under dark conditions at 27 ± 2°C until the embryo starts to germinate; after germination, sub - culture 2 - 3 times. Throughout the process, a high - sugar content of 60 g / L is used without reducing the sugar content; after rooting, dilute a commercial tissue - culture antifungal agent and soak the seedlings for 8 h. After washing once with a sugar - free Y3 medium, transfer them to a culture substrate. The composition of the culture substrate is commercial nutrient soil + vermiculite, with a composition of 50% nutrient soil and 50% vermiculite.

[0014] Preferably, the specific process of step S4 is: transferring the germinated embryo to a medium composed of Y3 liquid medium, 60 g / L of sucrose, and 2 g / L of activated carbon, using a 7:3 ratio of red - blue LED illumination, with a light intensity of 25 μmol m -2 s -1 , illuminating for 16 hours every day, and culturing for 8 - 12 weeks at 27 ± 1°C until the seedlings grow 1 - 2 leaves; or The specific process of step S5 is: selecting healthy tissue - cultured seedlings with a main root ≥5 cm and fibrous roots ≥20, placing them in a sugar - free Y3 liquid medium with 2 g / L of activated carbon added, controlling the CO 2 concentration to 1600 μmol / mol, using red - blue ratio of 7:3 and light intensity of 600 μmol m -2 s -1 LED illumination, illuminating for 16 hours every day for hardening off the seedlings.

[0015] Preferably, the specific process of step S6 is: in a laminar flow hood, completely wash the sugar - containing medium residue on the tissue - cultured seedlings with sterile water, then soak them in a 1 g / L carbendazim dilution solution, using 60 mL for each seedling for sterilization, and the sterilization time is 8 hours; after sterilization, transplant the tissue - cultured seedlings into disposable plastic cups filled with nutrient soil and vermiculite, and place them in an incubator; or The specific process of step S7 is: when the seedlings after hardening have more than 3 leaves and a strong root system, they are transplanted outdoors.

[0016] Compared with the prior art, this application has the following beneficial effects: (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 exogenous CO is added to the culture environment. 2 and different light qualities improved the growth traits and survival rate of embryo culture seedlings, which is beneficial to the promotion and cultivation of Macapuno coconut.

[0017] (2) The time required for this application is shortened, the time for the secondary roots to fully grow is reduced to 4-5 months, the survival rate of seedlings is increased to 94%, and the fungal infection rate is reduced to 17%. Compared with the traditional method, the fresh weight of seedlings, stem diameter and leaf width are 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

[0018] In order to more clearly illustrate the technical solutions in the present application or the prior art, the drawings required for use in the description of the present application or the prior art will be briefly introduced below. 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 paying any creative work.

[0019] Figure 1 This is a photo of embryo culture seedlings grown in continuous high-sugar tissue culture four months after embryo culture (left); compared with a photo of tissue culture seedlings grown in reduced-sugar tissue culture (right); the difference between the two in the root part is not much, but the root development is very different; Figure 2 For embryo culture seedlings of hardening 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 the top of the seedling covered with plastic sheeting to reduce water loss; 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

[0020] 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. Obviously, the described embodiments are part of the embodiments of this application, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in this application without creative efforts belong to the scope of protection of this application.

[0021] Unless otherwise specified, the experimental methods used in the embodiments of this application are all conventional methods.

[0022] In the following embodiments and comparative examples, unless otherwise specified, all raw materials can be obtained through commercial purchase or conventional methods.

[0023] 1. Material preparation ● Plant materials: Mature Marapuño coconut fruits (endosperm is semi-transparent and gelatinous).

[0024] ● Sterilization reagents: 2.4% NaClO solution, 70% ethanol, 0.8% NaClO solution, 1 g / L carbendazim dilution (commercial).

[0025] ● Culture medium: Based on the Y3 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.

[0026] Table 1 Composition of high-carbon culture medium Example 1

[0027] The high-carbon rooting method for Marapuño coconut tissue culture seedlings in this example includes the following steps: S1. Disinfection of embryo and tools: Use an embryo extractor to extract endosperm blocks from the coconut meat of Marapuño coconut, and locate the embryo position based on the large hole at the bottom of the coconut; Among them, the embryo extractor includes an embryo extraction tube with a length of 20 cm, an outer diameter of 3.5 cm, a wall thickness of 0.3 cm, made of steel, with the front end thinned to form a wedge shape for easy embedding into the endosperm, and a small stick at the rear. Pushing forward can push out the cylindrical endosperm block containing the embryo from the front end; Among them, when taking the embryo of Marapuño 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; Among them, spray the embryo extractor with 70% ethanol for disinfection before each extraction of the endosperm block.

[0028] S2. Disinfection treatment of endosperm blocks and embryos: Rinse the endosperm blocks with 2.4% NaOCl for 10 minutes. Then, cut the endosperm blocks in a laminar flow hood, take out the embryos, soak the embryos in 70% ethanol for 1 minute, then place them in 0.8% NaOCl for 10 minutes, and subsequently rinse them 5 times with sterile water, 1 minute each time. Finally, dry them in a laminar flow hood to obtain surface-sterilized zygotic embryos.

[0029] S3. Primary dark culture: 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. Throughout the process, use 60 g / L of high sugar without sugar reduction treatment. After rooting, dilute a commercial tissue culture antifungal agent and soak the seedlings for 8 h. After washing them once with sugar-free Y3 medium, transfer them to the culture substrate, and the composition of the culture substrate is commercial nutrient soil + vermiculite, with a composition of 50% nutrient soil and 50% vermiculite. Among them, the Y3 basal medium contains: Y3 macronutrients (mg / L): potassium nitrate KNO 3 2000.00, potassium chloride KCl 1500.00, ammonium chloride NH 4 Cl 530.00, sodium dihydrogen phosphate NaH 2 PO 4 ꞏ2H 2 O 310.00, calcium chloride CaCl 2 ꞏ2H 2 O 295.00, magnesium sulfate MgSO 4 ꞏ7H 2 O 250.00; Y3 micronutrients (mg / L): manganese sulfate MnSO 4 ꞏ4H 2 O 11.50, potassium iodide KI 8.00, zinc sulfate ZnSO 4 ꞏ7H 2 O 7.30, boric acid H 3 BO 3 3.00, copper sulfate CuSO 4 ꞏ5H 2 O 0.270, cobalt chloride CoCl 2 ꞏ6H 2 O 0.250, sodium molybdate NaMoO 4 ·H 2 O 0.250, nickel chloride NiClꞏ6H 2 O 0.026, ferrous sulfate Fe 2 SO4 ·7H 2 O 42.00, disodium ethylenediaminetetraacetate Na 2 EDTA 56.0; Vitamins and amino acids (mg / L): Pyridoxine hydrochloride (vitamin B6) C 8 H 11 NO 3 ·HCl 0.05, Thiamine hydrochloride (vitamin B1) C 12 H 17 ClN 4 OS·HCl 0.05, Niacin (vitamin B3) C 6 H 5 NO 2 0.05, Calcium pantothenate (vitamin B5) (C 9 H 16 NO 5 ) 2 Ca 0.05, Biotin (vitamin H) C 10 H 16 N 2 O 3 S 0.05, Folic acid (vitamin Bc, vitamin M) C 19 H 19 N 7 O 6 0.05, Glycine C 2 H 5 NO 2 1.00; Other additives: Abscisic acid (ABA(a)) C 15 H 20 O 4 0.25 mg / L, α-Naphthaleneacetic acid (NAA(b)) C 12 H 10 O 2 18.6mg / L, Sucrose C 6 H 22 O 11 50 - 60.0 g / L, Activated carbon 2.0 g / L; Among them, the whole process of tissue culture uses a sucrose content of 60g / L. The Y3 basal medium also contains 7.0 g / L of agar or 3.0 g / L of Gelzan. The pH value of the Y3 basal medium and the solid medium is 5.7.

[0030] S4, Seedling light culture: Transfer the germinated embryo to a medium composed of Y3 liquid medium, 60g / L sucrose and 2 g / L activated carbon, and use a 7:3 ratio of red and blue LED lighting with a light intensity of 25μmol m -2 s-1 Cultivate for 8 - 12 weeks at 27±1°C with 16 hours of light per day until the seedlings grow 1 - 2 leaves.

[0031] S5. Acclimatization treatment: Select healthy 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 CO 2 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 light per day for acclimatization.

[0032] S6. Preparation before transplantation: In a laminar flow hood, thoroughly wash the sugar - containing medium residue on the tissue culture seedlings with sterile water, then soak them in a 1 g / L carbendazim dilution, using 60 mL per 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.

[0033] S7. Transplantation: When the acclimatized seedlings have more than 3 leaves and strong roots, transplant them outdoors.

[0034] In this example, through full - process high - sugar culture (60 g / L sucrose), enhanced sterilization (soaking in carbendazim), and light - CO 2 co - regulation, the rooting period of Macapuno coconut tissue culture seedlings is significantly shortened to 4 - 5 months, the acclimatization survival rate is increased to 94%, and the fungal infection rate is reduced to 17%. Compared with the traditional method, the fresh weight, stem diameter, and leaf width of the seedlings are increased by 51.8%, 74%, and 44.9% respectively, with significant technical effects and the potential for industrial application.

[0035] Comparative Example 1 The technical solution of this comparative example refers to the prior art 202410635327.X.

[0036] Test Example 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.

[0037] Table 2

[0038] Table 3

[0039] Table 4

[0040] Compared with Comparative Example 1, the method of the present application has the following advantages: 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 ion exchange capacity, can slowly release nutrients, and reduce the initial topdressing frequency.

[0041] 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, with a water holding rate of 40 - 50%, significantly reducing the risk of drought stress.

[0042] 3. Disease resistance and root development optimization: The well-rotted 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.

[0043] The nutrient soil is rich in nitrogen, phosphorus, and potassium nutrients, and vermiculite has ion exchange capacity. The combination of the two can continuously supply fertilizer for tissue culture seedlings and reduce the topdressing frequency. The nutrient soil can regulate pH, and vermiculite can adsorb water and nutrients, reducing the drought stress risk of seedlings, with a water holding rate of 40 - 50%. Moreover, this combination can inhibit the reproduction of pathogenic bacteria and promote root development. Although its cost is 7.5 - 13 yuan / kg, which is higher than that of rice husk charcoal + coconut coir substrate, it can increase the root biomass by 10 - 15%.

[0044] At the same time, we found that the combination of rice husk charcoal + coconut coir are both organic substances. When contaminated, they are extremely prone 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 soaking in disinfectant, the risk is extremely high. While there is less organic matter in the nutrient soil and vermiculite, and the tissue culture seedlings soaked and sterilized hardly carry pathogenic bacteria, which can greatly improve the survival rate.

[0045] 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 the embryo culture seedlings acclimatized using 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 For the tissue culture seedlings that have successfully rooted in vitro (sterile) using the method of the present application, they 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 the present application have grown a large number of roots, greatly improving the survival rate.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than limiting them; although the present 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 described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A high carbon rooting method for Macapuno coconut tissue culture seedlings, characterized in that: The steps include: S1. Embryo extraction and tool disinfection: Use an embryo extractor to extract endosperm blocks from the coconut meat of the Macapuno coconut, and locate the embryo position according to the large hole at the bottom of the coconut; S2. Disinfection of endosperm blocks and embryos: rinse the endosperm blocks, cut the endosperm blocks in a clean bench, take out the embryos, soak the embryos, rinse them with sterile water, and finally blow dry them in a clean bench to obtain surface-sterilized zygote embryos; S3, primary dark culture: Place the surface-sterilized zygotic embryos on solid culture medium and cultivate them in the dark until the embryos begin to germinate; S4, seedling light culture: transfer the germinated embryos to a medium consisting of Y3 liquid medium, sucrose and activated carbon, and culture under light until the seedlings grow leaves; S5, seedling hardening treatment: Select robust tissue culture seedlings, place them in sugar-free Y3 liquid culture with activated carbon, and control the CO2 concentration and light intensity for seedling hardening; S6. Preparation before transplanting: In a clean bench, completely wash the culture medium residue with sugar on the tissue culture seedlings with sterile water, then soak them, and then transplant the tissue culture seedlings into a plastic disposable cup filled with nutrient soil and vermiculite, and put them into an incubator; S7. Transplanting: When the seedlings have leaves and strong roots after hardening, transplant them outdoors.

2. The high carbon rooting method of Macapuno coconut tissue culture seedlings according to claim 1, characterized in that: In step S1, the embryo extractor includes an embryo extractor tube, which is 20 cm long, 3.5 cm in outer diameter, 0.3 cm in wall thickness, made of steel, with the front end thinned to form a wedge shape for easy embedding into the endosperm, and a small stick at the rear, which can be pushed forward to push out the cylindrical endosperm block containing the embryo from the front end.

3. The high carbon rooting method of Macapuno coconut tissue culture seedlings according to claim 1, characterized in that: In step S1, when taking the embryo of the Macapuno coconut, the endosperm block containing the embryo is obtained by inserting the larger hole among the three holes at the bottom of the coconut into the corresponding position of the endosperm.

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

5. The high carbon rooting method of Macapuno coconut tissue culture seedlings according to claim 1, characterized in that: The specific process of step S2 is: rinse the endosperm block with 2.4% NaOCl for 10 minutes, then cut the endosperm block in the clean bench, take out the embryo, soak the embryo in 70% ethanol for 1 minute, and then put it in 0.8% NaOCl for 10 minutes, then rinse it with sterile water for 5 times, each time for 1 minute, and finally blow dry it in the clean bench.

6. The high carbon rooting method of Macapuno coconut tissue culture seedlings according to claim 1, characterized in that: In step S3, the Y3 liquid culture medium contains: Y3 Macronutrients: Potassium nitrate KNO3 2000.00mg / L, potassium chloride KCl 1500.00mg / L, ammonium chloride NH4Cl530.00mg / L, sodium dihydrogen orthophosphate NaH2PO4•2H2O 310.00mg / L, calcium chloride CaCl2•2H2O 295.00mg / L, magnesium sulfate MgSO4•7H2O 250.00mg / L; Y3 micronutrients: manganese sulfate MnSO4•4H2O 11.50mg / L, potassium iodide KI 8.00mg / L, zinc sulfate ZnSO4•7H2O 7.30mg / L, boric acid H3BO3 3.00mg / L, copper sulfate CuSO4•5H2O 0.270mg / L, cobalt chloride CoCl2•6H2O0.250mg / L, sodium molybdate NaMoO4•H2O 0.250mg / L, nickel chloride NiCl•6H2O 0.026mg / L, ferrous sulfate Fe2SO4•7H2O 42.00mg / L, disodium ethylenediaminetetraacetic acid Na2EDTA 56.0mg / L; Vitamins and amino acids: Pyridoxine hydrochloride C8H 11 NO3•HCl 0.05mg / L, Thiamine Hydrochloride C 12 H 17 ClN4OS•HCl 0.05mg / L, nicotinic acid C6H5NO2 0.05mg / L, calcium pantothenate (C9H 16 NO5) 2Ca 0.05mg / L, Biotin C 10 H 16 N2O3S0.05mg / L, folic acid C 19 H 19 N7O6 0.05mg / L, glycine C2H5NO2 1.00mg / L; Other additives: Abscisic acid (ABA(a)) C 15 H 20 O4 0.25 mg / L, α-naphthylacetic 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 of Macapuno coconut tissue culture seedlings according to claim 6, characterized in that: In step S3, a sucrose content of 60 g / L is used in the whole tissue culture process, the Y3 liquid culture medium also contains 7.0 g / L agar or 3.0 g / L Gelzan, and the pH value of the Y3 liquid culture medium and the solid culture medium is 5.

7.

8. The high carbon rooting method of Macapuno coconut tissue culture seedlings according to claim 1, characterized in that: The specific process of step S3 is: placing the surface-sterilized zygote embryo on a solid culture medium composed of Y3 liquid culture medium, 60g / L sucrose, 2g / L activated carbon, and 7g / L agar, and cultivating it in the dark at 27±2°C until the embryo begins to germinate; after germination, subculture 2-3 times, using 60g / L high sugar throughout the process without hypoglycemic treatment; after rooting, dilute with a commercial tissue culture antifungal agent, soak the seedlings for 8h, wash them once with sugar-free Y3 culture medium, and then transfer them to a culture matrix, the composition of the culture matrix is ​​commercial nutrient soil + vermiculite, with a composition of 50% nutrient soil and 50% vermiculite.

9. The high carbon rooting method of Macapuno coconut tissue culture seedlings according to claim 1, characterized in that: The specific process of step S4 is as follows: the germinated embryos are transferred to a medium consisting of Y3 liquid medium, 60 g / L sucrose and 2 g / L activated carbon, and illuminated by a 7:3 ratio of red and blue LEDs with a light intensity of 25 μmol m -2 s -1 , 16 hours of light per day, culture at 27±1℃ for 8-12 weeks, until the seedlings grow 1-2 leaves; or The specific process of step S5 is as follows: select robust tissue culture seedlings with a taproot ≥ 5 cm and fibrous roots ≥ 20, place them in a Y3 liquid medium without sugar and with 2 g / L activated carbon, control the CO2 concentration to 1600 μmol / mol, use a red-to-blue ratio of 7:3, and a light intensity of 600 μmol m -2 s -1 LED lighting, 16 hours of light per day for seedling hardening.

10. The high carbon rooting method for Macapuno coconut tissue culture seedlings according to claim 1, characterized in that: The specific process of step S6 is: in a clean bench, completely wash the culture medium residue with sugar on the tissue culture seedlings with sterile water, and then soak them in 1g / L carbendazim dilution, using 60mL for each seedling for sterilization, and the sterilization time is 8 hours; after sterilization, transplant the tissue culture seedlings into a plastic disposable cup filled with nutrient soil and vermiculite, and put them into an incubator; or The specific process of step S7 is: when the seedlings after hardening have more than 3 leaves and a strong root system, they are transplanted outdoors.

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