Tissue culture method for tarocalamus obtusifolius

Abstract

The invention discloses a method for tissue culture of taros nigra. The method comprises the following steps: treating an explant and carrying out inoculated culture; after the explants are inoculated and cultured to germinate and grow, cutting off buds and inoculating the buds into a starting induction culture medium to induce adventitious buds; cutting the block mass obtained by the induction culture in the previous step, and inoculating the cut block mass into a proliferation and propagation culture medium to culture a proliferation block mass seedling, wherein the proliferation and propagation culture medium comprises the following components: MS, 5mg / L BA, 30g / L sucrose and 6g / L carrageenan, and the pH value is 6.0-6.2; performing strong seedling transition: cutting the proliferated block mass seedlings obtained in the previous step, inoculating the cut proliferated block mass seedlings into a strong seedling transition culture medium, and culturing into seedlings; and rooting culture: cutting the transition seedling block mass obtained in the previous step, inoculating the cut transition seedling block mass into a rooting culture medium, and culturing until the tissue culture seedling qualified standard is reached. The method has the advantages that the proliferation rate of the tarocalamus oblongifolius can be increased to 2.0-2.5, the tissue culture seedlings are free of variation and withered and yellow leaves in the culture period, the qualification rate of the tissue culture seedlings is high, and the method is suitable for large-scale production and application.

Description

Technical Field

[0001] The invention relates to the technical field of tissue culture of Colocasia spp. plants, in particular to a tissue culture technology of Colocasia spp. Background Art

[0002] Black Diamond Elephant Ear Taro, Latin name Colocasia esculenta 'DiamondHead'. Araceae, Colocasia genus, distributed in Guangdong, Guangxi to southeastern Yunnan, China, and also in Southeast Asia. Figure 8 As shown, the petiole is smooth, purple to green, 19.1 cm long, 47.3 cm tall, 49.2 cm wide, 22.4 cm long, and 13.4 cm wide; the leaf surface is dark purple to dark green with purple veins, the back is green, halberd-shaped and oval, and the edges are wavy. Because of its unique appearance and ornamental value, it is favored by consumers in the market. However, because the black diamond elephant ear taro tends to grow seedlings during production and cultivation, the proliferation rate is low, and the leaves are prone to yellowing, which limits its large-scale production. Summary of the invention

[0003] The invention aims to provide a black diamond elephant ear taro tissue culture method to solve the problems of low proliferation rate, easy occurrence of yellow leaves, and limitation of large-scale production in the existing black diamond elephant ear taro tissue culture production process.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] A method for tissue culture of black diamond elephant ear taro, the process comprising:

[0006] (1) Explant treatment and inoculation culture;

[0007] (2) Induction: After the explants sprout and grow, the buds are cut and inoculated into the induction medium to induce adventitious buds;

[0008] (3) Propagation and expansion: the clumps obtained by the induction culture in the previous step are cut and inoculated into a proliferation and expansion medium to culture the proliferated clumps. The components of the proliferation and expansion medium are: MS, BA 5 mg / L, sucrose 30 g / L, carrageenan 6 g / L, and pH 6.0-6.2;

[0009] (4) Strong seedling transition: The proliferated seedlings obtained in the previous step are cut and inoculated into strong seedling transition medium to be cultured into seedlings. The transition seedlings that do not meet the seedling standards are cut into individual plants and then continue to be cultured into strong seedling transition medium until they are mature seedlings. The mature seedlings that have met the qualified standards for tissue culture seedlings can be directly shipped;

[0010] (5) Rooting culture: The transitional seedling clumps that have reached the seedling standard obtained in the previous step are cut and inoculated into the rooting medium and cultured until they reach the qualified standard of tissue culture seedlings.

[0011] Furthermore, the culture conditions for proliferation and expansion are: 25°C, 3000LX light, 12h light / 12h dark; culture period: 25-35 days.

[0012] Furthermore, the explant treatment includes pretreatment, disinfection,

[0013] The pretreatment process comprises: selecting a mother plant that is healthy, free of pests and diseases, and has pure genetic traits, and controlling the drying of the substrate; selecting a newly grown, young and tender terminal bud of the black diamond elephant ear taro bulb, cutting off the terminal bud above the substrate, wiping the surface of the terminal bud with alcohol, cutting off the leaf horizontally, exposing the internal stem segment, and then cutting off the next leaf segment, wiping the internal stains with alcohol cotton after each leaf is taken off, and then girdling the next leaf until the leaf cannot be divided again, and wiping the surface of the explant with alcohol to obtain the explant;

[0014] The disinfection process includes: first disinfecting the explant with 75% alcohol for 10-30 seconds, then disinfecting with 0.1% mercuric chloride for 20-25 minutes, and then rinsing with sterile water after the disinfection is completed.

[0015] Furthermore, the inoculation operation process includes: using left-hand tweezers to tightly clamp the stem segment of the explant, scraping off a layer of epidermis from the wound that contacts the disinfectant, cutting off a 1-2 mm length portion from the base, clamping the entire explant with tweezers, and inoculating it into the culture medium with the bud point facing upward;

[0016] The explant inoculation culture medium comprises MS, 30 g / L sucrose, 6 g / L carrageenan, and a pH value of 6.0-6.2;

[0017] Culture conditions: 25°C, 3000LX light, 12h light / 12h dark;

[0018] Cultivation period: 24-30 days.

[0019] Furthermore, the induction process includes: when the explants are inoculated and cultured to germinate lateral buds and terminal buds and grow to 2-3 cm, the new buds are cut off along the edge of the explants and the surrounding brown leaves are cut off, the overlong terminal buds are removed, and then inoculated into the induction medium. If adventitious buds cannot be induced in one induction, a second induction can be performed;

[0020] The components of the start-up induction medium are: MS, BA 3-5 mg / L, sucrose 30 g / L, carrageenan 6 g / L, pH 6.0-6.2;

[0021] Culture conditions: 25°C, 3000LX light, 12h light / 12h dark;

[0022] Cultivation period: 30-35 days.

[0023] Furthermore, the seedling-strengthening transition process includes: dividing the proliferation mass seedlings into 2-3 bud masses or large bud single seedlings, cutting off the withered leaves at the base, removing the tops, cutting off the grown roots, and inoculating them into a seedling-strengthening transition medium for cultivation;

[0024] The composition of the seedling transition medium is: MS, paclobutrazol 0.5-0.8 mg / L, sucrose 30 g / L, carrageenan 6 g / L, pH 6.0-6.2;

[0025] Culture conditions: 25°C, 3000LX light, 12h light / 12h dark;

[0026] Cultivation period: 20-25 days.

[0027] Furthermore, the rooting culture process includes: selecting transitional seedling clumps that meet the seedling standard, cutting the clumps into individual plants without removing the tops, cutting off the root system and withered and rotten leaves of the clumps, and inoculating the individual plants into a rooting medium for rooting culture;

[0028] Culture conditions: 25°C, 3000LX light, 12h light / 12h dark;

[0029] Cultivation period: 7-10 days.

[0030] Furthermore, the rooting medium comprises the following ingredients: MS, 30 g / L sucrose, 6 g / L carrageenan, and a pH value of 6.0-6.2.

[0031] Furthermore, the rooting medium comprises the following ingredients: MS, AC 0.1-0.3 g / L, sucrose 30 g / L, carrageenan 6 g / L, and a pH value of 6.0-6.2.

[0032] Furthermore, the seedling standard is: plant height 3-4 cm, more than 2 leaves, and leaves are stretched;

[0033] Qualified standards for tissue culture seedlings: plant height 4-5cm, crown width 4-6cm, leaf length 2-3cm, leaf width 1.5-2.5cm, more than 3 leaves, root length 3-4cm, and more than 5 roots.

[0034] The invention has the advantages that the proliferation rate of the black diamond elephant ear taro can be increased to 2.0-2.5, no mutation occurs in the tissue culture seedlings during the culture period, no withered and yellow leaves appear, the qualified rate of the tissue culture seedlings is high, and the invention is suitable for large-scale production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The drawings described herein are used to provide a further understanding of the present invention, constitute a part of the present application, and do not constitute an improper limitation of the present invention. In the drawings:

[0036] Figure 1 It is a schematic diagram of the states of explants at different treatment stages;

[0037] Figure 2 These are photos of explants from the terminal bud of the bulb at the time of inoculation and 24 days after inoculation;

[0038] Figure 3 These are photos taken when the induction inoculation was initiated and on day 32 of culture;

[0039] Figure 4 This is a photo of the multiplication and expansion inoculation and culture for 34 days;

[0040] Figure 5 This is a photo of the strong seedlings being inoculated and cultured for 20 days;

[0041] Figure 6 It is a photo of qualified standards for tissue culture seedlings;

[0042] Figure 7 These are photos of rooting culture inoculation and cultivation for 7 days;

[0043] Figure 8 It is a photo of the Black Diamond Elephant Ear Taro;

[0044] Fig. 9 This is a photo of the tissue culture seedlings cultivated in Example 2;

[0045] Fig.10 This is a photo of Example 3 Treatment 1 after inoculation and cultivation;

[0046] Fig.11 This is a photo of Example 3 after treatment 2 inoculation and cultivation;

[0047] Fig.12 This is a photo of Example 3 after inoculation and cultivation of Treatment 3;

[0048] Fig.13 This is a photo of Example 3 after treatment 4 inoculation and cultivation;

[0049] Fig.14 This is a photo of Example 4 after treatment 1 inoculation and cultivation;

[0050] Fig.15 This is a photo of Example 4 after treatment 2 inoculation and cultivation;

[0051] Fig.16 This is a photo of Example 4 after treatment 3 inoculation and cultivation;

[0052] Fig.17 This is a photograph of Example 4 after treatment 4 was inoculated and cultured. DETAILED DESCRIPTION

[0053] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. The exemplary embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0054] Example 1

[0055] This embodiment provides a method for tissue culture of black diamond elephant ear taro, and the specific process is as follows:

[0056] STG0 maternal pretreatment:

[0057] Select mother plants that are healthy, free of pests and diseases, and have pure genetic traits, and remove the drying medium two days in advance.

[0058] Sterilization and inoculation of STG1 explants:

[0059] ① Explant pretreatment

[0060] a) Time: Sunny day;

[0061] b) Processing tools: scissors, sharp scalpels disinfected with 75% alcohol, alcohol cotton;

[0062] c) Treatment method: Try to select newly grown, tender apical buds of the black diamond elephant ear taro bulbs. Cut off the apical buds above the substrate, discard the bottom stem section that touches the substrate, and clean the remaining dirt on the surface. After cleaning, place the cut apical buds on a plate.

[0063] ② Explant fine processing

[0064] a) Handling tools: Disinfect sharp scalpels with 75% alcohol;

[0065] b) Treatment method: First, disinfect your hands and the knives you use with 75% alcohol; wipe the surface of the apical bud clean with alcohol cotton. Figure 1 As shown in the figure above, after wiping it clean, use a scalpel to cut off the leaves in a ring, expose the internal stem segment, and then cut off the next segment of leaves in a ring again. After each leaf is removed, wipe the internal stains with alcohol cotton, and then cut off the next leaf again until the leaf cannot be divided again. Finally, wipe the surface of the explant again with alcohol cotton to obtain an explant with a diameter of 1.5 cm and a length of 2.2 cm. Figure 1 As shown in the lower left picture.

[0066] ③ Explant disinfection

[0067] a) Preparation before disinfection: turn on the lighting and disinfection furnace to disinfect the inoculation tools, use 75% alcohol to disinfect the scalpel, wipe the clean bench and disinfect your hands;

[0068] b) Processing tools: 75% alcohol, mercuric chloride, 6 bottles of sterile water, sterilized wide-mouth bottles, and bottles for waste mercury;

[0069] c) Disinfection method: First disinfect the explants with 75% alcohol for 10-30 seconds, then disinfect with 0.1% mercuric chloride for 20-25 minutes. The disinfection time of mercuric chloride should be adjusted according to the size and tenderness of the stem segment. After disinfection, rinse with sterile water 5-6 times.

[0070] ④ Sterile inoculation

[0071] a) Processing tools: culture medium, sterilized inoculation tools.

[0072] b) Treatment method: Use left-hand tweezers to firmly grasp the stem segment of the explant, and remove a layer of epidermis from the wound that has come into contact with the disinfectant. Cut off a 1-2 mm long section at the base. Figure 1 As shown in the lower right picture; use tweezers to hold the entire explant, insert it into the culture medium with the bud point facing upwards, the insertion depth of the culture medium is about 3mm, and one explant is inoculated in each bottle. Figure 2 The left picture shows the inoculation of the explant from the bulb apical bud. Be careful not to clamp the bud with the tweezers during inoculation, which may damage or kill the bud. Fill out the start-up record sheet after inoculation.

[0073] c) Inoculation medium: MS, sucrose 30 g / L, carrageenan 6 g / L, pH 6.0-6.2.

[0074] d) Culture conditions: 25°C, 3000LX light, 12h light / 12h dark.

[0075] Note: After one week, pick out those contaminated by fungi and bacteria; check frequently to observe the growth of the explants.

[0076] 24 days after inoculation, the growth status of the explants from the bulb apical buds is as follows Figure 2 As shown in the right picture, each explant was successfully inoculated and cultured, so the explant sterilization success rate was 100%.

[0077] STG2 starts the induction period

[0078] When the lateral buds and terminal buds germinate and grow to about 2-3cm 30 days after inoculation, cut off the new buds along the edge of the explant and remove the surrounding brown leaves, remove the top of the terminal buds that are too long, and then inoculate them into the start-up induction medium. If adventitious buds cannot be induced in one induction, a second induction can be performed.

[0079] a) Start induction medium: MS, BA 3mg / L, sucrose 30g / L, carrageenan 6g / L, pH 6.0-6.2, 1 bud / bottle.

[0080] b) Culture conditions: 25°C, 3000LX light, 12h light / 12h dark.

[0081] c) Culture period: 30-35 days.

[0082] like Figure 3 The left picture shows the vaccination photo. Figure 3 The right picture shows the photograph after 32 days of induction culture, and the bud induction rate is 100%.

[0083] STG3 proliferation and expansion period

[0084] When the number of clumps reaches a certain amount, large-scale propagation will be carried out. The cutting requirements are: divide the propagation seedling clumps into small ones, transfer the clumps with small buds of 2-3 buds, remove the tops, cut off the dead leaves and roots at the base, and the large bud single plant can be broken from the middle but not completely cut off for inoculation. If the diameter of the large bud single plant is greater than 0.8cm, it can be completely cut in half for inoculation.

[0085] a) Proliferation and expansion medium: MS, BA 5mg / L, sucrose 30g / L, carrageenan 6g / L, pH 6.0-6.2, 10 pellets / bottle.

[0086] b) Culture conditions: 25°C, 3000LX light, 12h light / 12h dark.

[0087] c) Culture period: 25-35 days.

[0088] Photos of vaccinations Figure 4 As shown in the left picture, the photo of the proliferation and expansion culture on day 34 is as follows Figure 4 As shown in the right picture, there is no yellowing of leaves, and the proliferation rate is 2.0-2.5. Variation: No variation occurred in the tissue culture seedlings during the culture period.

[0089] STG4 seedling transition period

[0090] Cutting requirements: Divide the proliferation cluster seedlings into 2-3 bud clusters or large bud single seedlings, cut off the withered leaves at the base, remove the top, and cut off the grown roots.

[0091] a) Seedling transition medium: MS, paclobutrazol (pp333) 0.5 mg / L, sucrose 30 g / L, carrageenan 6 g / L, pH 6.0-6.2, 8 balls / bottle. Use high wide-mouth bottles for inoculation and cultivation.

[0092] b) Culture conditions: 25°C, 3000LX light, 12h light / 12h dark.

[0093] c) Culture period: 20-25 days.

[0094] Photos of vaccinations Figure 5 As shown in the left picture, the photo of the strong seedlings after 20 days of transitional cultivation is as follows Figure 5As shown in the right picture, there was no variation in the transition seedlings during the cultivation period. The plants were strong and the dwarfing effect was good. Seedling standard: plant height 3-4cm, more than 2 leaves, and the leaves were stretched. The seedling rate after 20 days of cultivation was 88.3%. Figure 6 As shown in the figure, the standard for a single plant of tissue culture seedlings is: plant height 4-5cm, crown width 4-6cm, leaf length 2-3cm, leaf width 1.5-2.5cm, more than 3 leaves, root length 3-4cm, and more than 5 roots. After the transitional cultivation of strong seedlings, the qualified seedlings in the mass are cut off for rooting. If the seedlings have reached the qualified standards of tissue culture seedlings, they can be shipped directly. The transitional seedlings that do not meet the standards are cut into single plants and then continue to be cultured for strong seedlings.

[0095] STG5 rooting period

[0096] Cutting requirements: Select transitional seedling clumps that meet the seedling standards, cut the clumps into individual plants without removing the tops, and cut off the roots and withered and rotten leaves of the clumps, and inoculate the individual plants into rooting medium 1 or rooting medium 2 for rooting culture:

[0097] a) Rooting medium 1: MS, sucrose 30 g / L, carrageenan 6 g / L, pH 6.0-6.2;

[0098] Rooting medium 2: MS, AC 0.2g / L, sucrose 30g / L, carrageenan 6g / L, pH 6.0-6.2; 8-10 plants / bottle.

[0099] b) Culture conditions: 25°C, 3000LX light, 12h light / 12h dark.

[0100] c) Culture period: 7-10 days.

[0101] During the rooting and cultivation period, the root growth should be observed and adjustments should be made to prevent the subsequent roots from growing too long and affecting the screening and planting of seedlings.

[0102] When inoculated into rooting medium 1, the photo is as follows Figure 7 As shown in the upper left picture, the photo of rooting culture at 7 days Figure 7 As shown in the lower left picture, the photo is as follows when inoculated into rooting medium 2 Figure 7 As shown in the upper right picture, the photo of rooting culture at 7 days Figure 7 As shown in the lower right figure, no mutation occurred during the culture period.

[0103] Example 2

[0104] This example provides different induction culture media for inducing adventitious bud growth test. The induction culture media of this example includes MS, BA 2 mg / L, sucrose 30 g / L, carrageenan 6 g / L, and pH 6.0-6.2.

[0105] Photos after induction culture Fig. 9As shown, no adventitious buds grew during the culture period, and the growth of tissue culture seedlings was more obvious.

[0106] Example 3

[0107] This example provides different proliferation and expansion culture media for proliferation rate test, as shown in Table 1 below:

[0108] Table 1

[0109]

[0110] In addition to the components shown in Table 1, the components of each culture medium also include 30 g / L sucrose, 6 g / L carrageenan, and a pH value of 6.0-6.2.

[0111] The inoculation photos and the photos after cultivation of treatment 1 are as follows Fig.10 As shown, the inoculation photos and the photos after cultivation of treatment 2 are as follows Fig.11 The inoculation photos and the photos after cultivation of treatment 3 are shown in Fig.12 As shown, the inoculation photos and the photos after cultivation of treatment 4 are shown in Fig.13 shown.

[0112] From Table 1, Figure 10-13 It can be seen that the proliferation rates of the four treatments were not high, and the leaves all showed yellowing.

[0113] Example 4

[0114] This example provides different seedling-strengthening transition culture media for seedling-strengthening test, as shown in Table 2:

[0115] Table 2

[0116]

[0117] In addition to the components shown in Table 2, the components of each culture medium also include 30 g / L sucrose, 6 g / L carrageenan, and a pH value of 6.0-6.2.

[0118] Figure 14-17 As shown, these are photos of treatments 1 to 4 at the time of inoculation and 20 days of culture. Treatment 1 had the highest seedling rate and the shortest seedling time, but the plants were elongated; Treatments 2 and 3 added low concentrations of pp333, the dwarfing effect was average, and the plant type was tall; Treatment 4 added high concentrations of pp333, the dwarfing effect was good, and the growth of the clump seedlings was good; no yellow leaves or mutations were found during the culture period.

[0119] The technical solutions provided by the embodiments of the present invention are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present invention. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, according to the embodiments of the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for tissue culture of black diamond elephant ear taro, characterized in that: The process includes: (1) Explant treatment and inoculation culture; (2) Induction: After the explants sprout and grow, the buds are cut and inoculated into the induction medium to induce adventitious buds; (3) Propagation and expansion: the clumps obtained by the induction culture in the previous step are cut and inoculated into a proliferation and expansion medium to culture the proliferated clumps. The components of the proliferation and expansion medium are: MS, BA 5 mg / L, sucrose 30 g / L, carrageenan 6 g / L, and pH 6.0-6.2; (4) Strong seedling transition: The proliferated seedlings obtained in the previous step are cut and inoculated into strong seedling transition medium to be cultured into seedlings. The transition seedlings that do not meet the seedling standards are cut into individual plants and then continue to be cultured into strong seedling transition medium until they are mature seedlings. The mature seedlings that have met the qualified standards for tissue culture seedlings can be directly shipped; (5) Rooting culture: The transitional seedling clumps that have reached the seedling standard obtained in the previous step are cut and inoculated into the rooting medium and cultured until they reach the qualified standard of tissue culture seedlings.

2. A method for tissue culture of the black diamond elephant ear taro according to claim 1, characterized in that: The culture conditions for proliferation and expansion are: 25° C., 3000 LX of light, 12 h light / 12 h dark; and culture period: 25-35 days.

3. A method for tissue culture of the black diamond elephant ear taro according to claim 1 or 2, characterized in that: The explant treatment includes pretreatment and disinfection. The pretreatment process comprises: selecting a mother plant that is healthy, free of pests and diseases, and has pure genetic traits, and controlling the drying of the substrate; selecting a newly grown, young and tender terminal bud of the black diamond elephant ear taro bulb, cutting off the terminal bud above the substrate, wiping the surface of the terminal bud with alcohol, cutting off the leaf horizontally, exposing the internal stem segment, and then cutting off the next leaf segment, wiping the internal stains with alcohol cotton after each leaf is taken off, and then girdling the next leaf until the leaf cannot be divided again, and wiping the surface of the explant with alcohol to obtain the explant; The disinfection process includes: first disinfecting the explant with 75% alcohol for 10-30 seconds, then disinfecting with 0.1% mercuric chloride for 20-25 minutes, and then rinsing with sterile water after the disinfection is completed.

4. A method for tissue culture of the black diamond elephant ear taro according to claim 1 or 2, characterized in that: The inoculation operation process comprises: using left-hand tweezers to tightly clamp the stem segment of the explant, scraping off a layer of epidermis from the wound that contacts the disinfectant, cutting off a 1-2 mm length portion from the base, clamping the entire explant with tweezers, and inoculating it into the culture medium with the bud point facing upward; The explant inoculation culture medium comprises MS, 30 g / L sucrose, 6 g / L carrageenan, and a pH value of 6.0-6.2; Culture conditions: 25°C, 3000LX light, 12h light / 12h dark; Cultivation period: 24-30 days.

5. A method for tissue culture of the black diamond elephant ear taro according to claim 1, characterized in that: The induction process includes: when the explants are inoculated and cultured to germinate lateral buds and terminal buds and grow to 2-3 cm, the new buds are cut off along the edge of the explants and the surrounding brown leaves are cut off, the overlong terminal buds are removed, and then inoculated into the induction medium. If the adventitious buds cannot be induced in one induction, a second induction can be performed; The components of the start-up induction medium are: MS, BA 3-5 mg / L, sucrose 30 g / L, carrageenan 6 g / L, pH 6.0-6.2; Culture conditions: 25°C, 3000LX light, 12h light / 12h dark; Cultivation period: 30-35 days.

6. A method for tissue culture of the black diamond elephant ear taro according to claim 1, characterized in that: The seedling-strengthening transition process includes: dividing the proliferation mass seedlings into 2-3 bud masses or large bud single seedlings, cutting off the withered leaves at the base, removing the tops, cutting off the grown roots, and inoculating them into a seedling-strengthening transition medium for cultivation; The composition of the seedling transition medium is: MS, paclobutrazol 0.5-0.8 mg / L, sucrose 30 g / L, carrageenan 6 g / L, pH 6.0-6.2; Culture conditions: 25°C, 3000LX light, 12h light / 12h dark; Cultivation period: 20-25 days.

7. A method for tissue culture of the black diamond elephant ear taro according to claim 1, characterized in that: The rooting culture process comprises: selecting transitional seedling clumps that meet the seedling standard, cutting the clumps into individual plants without removing the tops, cutting off the root system and withered and rotten leaves of the clumps, and inoculating the individual plants into a rooting medium for rooting culture; Culture conditions: 25°C, 3000LX light, 12h light / 12h dark; Cultivation period: 7-10 days.

8. A method for tissue culture of the black diamond elephant ear taro according to claim 7, characterized in that: The rooting medium comprises the following components: MS, 30 g / L sucrose, 6 g / L carrageenan, and a pH value of 6.0-6.

2.

9. A method for tissue culture of the black diamond elephant ear taro according to claim 7, characterized in that: The rooting medium comprises the following components: MS, AC 0.1-0.3 g / L, sucrose 30 g / L, carrageenan 6 g / L, and a pH value of 6.0-6.

2.

10. The method for tissue culture of the black diamond elephant ear taro according to claim 1, characterized in that: The seedling standard is: plant height 3-4 cm, more than 2 leaves, and leaves are stretched; Qualified standards for tissue culture seedlings: plant height 4-5cm, crown width 4-6cm, leaf length 2-3cm, leaf width 1.5-2.5cm, more than 3 leaves, root length 3-4cm, and more than 5 roots.

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