CN115380827A - A kind of tissue culture rapid propagation method of green rainbow arrowroot - Google Patents

Abstract

The invention discloses a tissue culture rapid breeding method of green rainbow arrowroot, which comprises the following steps of S01: before the explant is taken from the female parent, performing drenching treatment, cutting robust lateral buds from the female parent, soaking, washing and drying the cut, flattening the cut and cutting for later use; s02: performing adventitious bud induction culture on the treated explant until adventitious bud plantlets are obtained; s03: dividing adventitious bud plantlets into single plants, reserving stem segments, inoculating the single plants into an adventitious bud multiplication culture medium for culture, and performing subculture multiplication until a plurality of strong adventitious buds are obtained; s04: cutting the strong adventitious bud into single plantlet, inoculating in rooting culture medium, and culturing to obtain complete plantlet with main root; s05: taking out the whole plant plantlet from the culture bottle, transferring to a water curtain greenhouse, planting in a 72-hole tray, spraying chlorothalonil after planting, and spraying manure water after growing a first new leaf. The invention can achieve the purposes of rapidly propagating the green rainbow arrowroot seedlings, reducing the pollution rate of seed production and improving the seedling rate.

Description

A kind of tissue culture rapid propagation method of green rainbow arrowroot

technical field

The invention relates to the technical field of green rainbow arrowroot breeding, in particular to a rapid tissue culture breeding method for green rainbow arrowroot.

Background technique

Green rainbow arrowroot Arrowroot is a perennial herbaceous foliage plant. Native to Brazil. This kind of leaf color is rich and colorful, with strong ornamental value, and most of them are shade plants with strong shade tolerance. It is an excellent indoor shade-loving foliage plant, suitable for decorating bedrooms, living rooms, offices and other places.

Yet fail to satisfy the great demand of market to green rainbow arrowroot at home, its main reason is that green rainbow arrowroot is hard to form seeds, and tillering ability is poor, and growth cycle is long, is difficult to propagate a large amount of finished products rapidly.

Therefore the present application proposes a tissue culture rapid breeding method of green rainbow arrowroot and utilizes tissue culture technology to rapidly and massively breed green rainbow arrowroot seedlings to solve the market demand.

Contents of the invention

The object of the present invention is to provide a kind of tissue culture rapid propagation method of green rainbow arrowroot, to solve the problems raised in the above-mentioned background technology.

Technical scheme of the present invention is realized like this:

A tissue culture rapid breeding method of green rainbow arrowroot, comprising:

S01: Before taking the explants from the female parent, perform irrigation treatment, cut the strong lateral buds from the female parent, soak, rinse and dry the incision, after ozone fumigation, then soak in hydrogen peroxide for 1-2 hours, and sterile water Rinse, sway and soak the explants in alcohol, rinse with sterile water, sway and soak in sodium hypochlorite aqueous solution, rinse with sterile water, smooth out the incision and remove it for later use;

S02: Inducing and culturing the treated explants for adventitious buds until seedlings with adventitious buds are obtained;

S03: Divide the adventitious bud seedlings into individual plants, remove the leaves, keep the stem section, inoculate into the adventitious bud proliferation medium for cultivation, and subculture the new buds and original buds until multiple robust adventitious buds are obtained. The adventitious buds are multiplied;

S04: cut the vigorously propagated adventitious buds into individual seedlings, inoculate them in the rooting medium for cultivation, and obtain complete plant seedlings with taproots;

S05: Take out the complete plant seedlings from the culture bottle, wash off the medium, transfer to the water curtain greenhouse, and plant them in the 72-hole tray. After planting, they are sprayed with chlorothalonil, and after the first new leaf grows, they are sprayed with fertilizer and water , the survival rate was 95%.

Adding low-concentration thiadizuron to the formulation of adventitious bud induction culture and adventitious bud proliferation medium helps to promote bud differentiation in the process of proliferation, and at the same time promotes bud expansion, increasing the proliferation coefficient and survival rate of buds.

Add ascorbic acid to the culture medium of adventitious bud induction culture, proliferation culture of adventitious buds, and rooting culture to inhibit the accumulation of quinones in the culture medium, reduce the poison of quinones to the seedlings of proliferating buds, and improve the efficiency and efficiency of value-added buds to absorb nutrients. Improve the survival rate of rooted seedlings.

Adding a low concentration of 6-benzylaminopurine to the rooting medium can make the plant grow robustly, increase the chlorophyll content of the leaves, and brighten the leaf color. The ambient temperature is 24-26°C, the light duration is 10 hours, and the light intensity is 1500-2500lux. It can reproduce stably for a long time Strong tissue culture seedlings are produced, the rooting rate is over 95%, and the root system is healthy.

Preferably, in the S01 step, the female parent is treated with 1500 times of chlorothalonil before taking the explants, the temperature of the custody environment is kept at 20-28°C, the humidity is kept at 80-95%, and the light intensity is kept at 5000-10000lux. Wait for the peat humidity of the female parent to drop to 18-20%, cut off 3-5cm long robust side buds from the female parent, peel off the old leaf sheaths, keep the innermost 2-3 layers of clean leaf sheaths, cut the incision flat with a scalpel, and clean the water Add detergent and soak for 5-10 minutes, rinse with water and dry, fumigate with ozone for 40-60 minutes, soak in 5% hydrogen peroxide for 1-2 hours, rinse with sterile water twice. Transfer the treated explants to the ultra-clean workbench, sway and soak them with 75% alcohol for 40-70s, rinse once with sterile water, put them in 0.6% sodium hypochlorite aqueous solution, sway and soak for 20-30 minutes, ensure that The explants were in full contact with the disinfectant solution, rinsed with sterile water 3 times, the outermost layer of leaf sheath was peeled off, the incision was leveled, and the injured tissue after disinfection was excised for later use.

Preferably, in the S02 step, the treated explants are inoculated into the adventitious bud induction medium for cultivation, and the adventitious bud seedlings can be obtained after culturing for 50-60 days, the culture environment temperature is 24-26°C, and the light duration is 10 hours. Light intensity 800-1500lux.

Preferably, in the S03 step, the adventitious bud seedlings are divided into individual plants, the leaves are removed, and the stem section 1cm is reserved, which is inoculated into the adventitious bud proliferation medium for cultivation. After 50-60 days of cultivation, the stem section re-grows 2- 3 seedlings with a multiplication coefficient of 2.0-3.0. Divide the grown adventitious bud seedlings into individual plants, remove the leaves, keep the stem section 1-1.5 cm, inoculate into the adventitious bud medium to continue the next generation culture, and the total number of subcultures is 15- 20 times to obtain robust adventitious bud seedlings, the cultivation environment temperature is 24-26°C, the light duration is 10 hours, and the light intensity is 1500-2500lux.

Preferably, in the S04 step, the vigorously propagated adventitious buds are cut into individual seedlings, inoculated in the rooting medium for cultivation, and after 30-40 days, a complete plant with 2-3 main roots can be obtained, and the rooting rate reaches 95%. -98%, the culture environment temperature is 24-26°C, the light duration is 10 hours, and the light intensity is 1500-2500lux. .

Preferably, in the S05 step, the complete plant seedlings are taken out from the culture bottle, the culture medium is washed off with clean water, transferred to a water curtain greenhouse, planted in a 72-hole tray, and sprayed once with 2000 times of bacterium Qing, the humidity of the planting environment is 80-95%, the temperature is 26-30°C, and the substrate for planting is peat:coconut peat=4:1. The thickness of the peat is 5-25. After the first new leaf grows, it is poured with fertilizer and water, and the survival rate reaches 95%.

Preferably, the medium selected for the adventitious bud induction culture in the step S02, the proliferation culture for the adventitious buds in the step S03, and the rooting culture in the step S04 includes: NH ₄ NO ₃ 990 mg/L, KNO ₃ 1140 mg/L, MgSO ₄ 7H ₂ O2 22mg/L, KH ₂ PO ₄ 102mg/L, CaCl ₂ 2H ₂ O2 64mg/L, KI0.83mg/L, H ₃ BO ₃ 6.2mg/L, MnSO ₄ 4H ₂ O2 2.3mg/L L, ZnSO ₄ 7H ₂ O 8.6mg/L, Na ₂ MoO ₂ 2H ₂ O 0.25mg/L, CuSO ₄ 5H ₂ O 0.025mg/L, CoCl ₂ 6H ₂ O 0.025mg/L, FeSO ₄ 7H ₂ O 27.8mg/L, Na ₂ EDTA 37.3mg/L, thiamine hydrochloride 0.1mg/L, niacin 0.5mg/L, pyridoxine hydrochloride 0.5mg/L, glycine 2mg/L, ascorbic acid 5 -10mg/L, inositol 100mg/L.

Preferably, in the S02 step, the induction medium is selected when explants are subjected to adventitious bud induction culture, and the induction medium also includes white sugar 30g/L, carrageenan 8.0g/L, 6-benzylaminopurine 1.0-4.0 mg/L, Thidiazuron 0.01-0.08mg/L, Naphthaleneacetic acid 0.05-0.2mg/L.

Preferably, in the S03 step, when the adventitious buds are proliferated, the proliferation medium is selected, and the proliferation medium also includes white sugar 30g/L, carrageenan 8.0g/L, 6-benzylaminopurine 0.5-2.0mg/L L, thiadiuron 0.01-0.08mg/L, indole butyric acid 0.1-0.2mg/L.

Preferably, in the S04 step, a rooting medium is used when rooting a single seedling, and the rooting medium also includes 25 g/L of white sugar, 8.0 g/L of carrageenan, and 0.1-0.2 mg of 6-benzylaminopurine /L, naphthalene acetic acid 0.01-0.02mg/L, indole butyric acid 0.1-0.4mg/L, activated carbon 0.2g/L.

Compared with prior art, the beneficial effect of the present invention is:

Adding low-concentration thiadizuron to the formulation of adventitious bud induction culture and adventitious bud proliferation medium helps to promote bud differentiation in the process of proliferation, and at the same time promote bud expansion, which improves the proliferation coefficient and survival rate of buds;

Add ascorbic acid to the culture medium of adventitious bud induction culture, proliferation culture of adventitious buds, and rooting culture to inhibit the accumulation of quinones in the culture medium, reduce the poison of quinones to the seedlings of proliferating buds, and improve the efficiency and efficiency of value-added buds to absorb nutrients. Improve the survival rate of rooted seedlings;

Adding a low concentration of 6-benzylaminopurine to the rooting medium can make the plant grow robustly, increase the chlorophyll content of the leaves, and brighten the leaf color. The ambient temperature is 24-26°C, the light duration is 10 hours, and the light intensity is 1500-2500lux. It can reproduce stably for a long time Strong tissue culture seedlings are produced, the rooting rate is over 95%, and the root system is healthy;

In the transplanting step of tissue culture seedlings, the substrate for planting is peat: coir peat = 4:1, which greatly improves the survival rate of tissue culture seedlings after colonization, and can make the survival rate of tissue culture seedlings after colonization reach 95%.

Description of drawings

Fig. 1 is the schematic flow sheet of the tissue culture rapid propagation method of a kind of green rainbow arrowroot proposed by the present invention.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1

A tissue culture rapid breeding method of green rainbow arrowroot, comprising:

S01: Before taking the explants from the female parent, perform irrigation treatment, cut the healthy lateral buds from the female parent, soak, rinse and dry the incision, after ozone fumigation, then shake and soak in hydrogen peroxide for 1 hour, rinse with sterile water, Shake and soak the explants in alcohol, rinse with sterile water, shake and soak in sodium hypochlorite aqueous solution, rinse with sterile water, smooth the incision and remove it for later use;

S02: Inducing and culturing the treated explants for adventitious buds until seedlings with adventitious buds are obtained;

S03: Divide the adventitious bud seedlings into individual plants, remove the leaves, keep the stem section, inoculate into the adventitious bud proliferation medium for cultivation, and subculture the new buds and original buds until multiple robust adventitious buds are obtained. The adventitious buds are multiplied;

S04: cut the vigorously propagated adventitious buds into individual seedlings, inoculate them in the rooting medium for cultivation, and obtain complete plant seedlings with taproots;

S05: Take out the complete plant seedlings from the culture bottle, wash off the medium, transfer to the water curtain greenhouse, and plant them in the 72-hole tray. After planting, they are sprayed with chlorothalonil, and after the first new leaf grows, they are sprayed with fertilizer and water , the survival rate was 95%.

In the present embodiment, in the step S01, the female parent was treated with 1500 times of chlorothalonil before taking the explants, the temperature of the custody environment was kept at 20°C, the humidity was kept at 80%, and the light intensity was kept at 5000 lux. Wait for the peat humidity of the female parent to drop to 18%, cut off 3-5cm long healthy lateral buds from the female parent, peel off the old leaf sheaths, keep the innermost 2-3 layers of clean leaf sheaths, cut the incision flat with a scalpel, add water to wash Soak in detergent for 5 minutes, rinse with water and dry, fumigate with ozone for 40 minutes, soak in 5% hydrogen peroxide for 1 hour, rinse with sterile water twice. Transfer the treated explants to the ultra-clean workbench, shake and soak them with 75% alcohol for 40 seconds, rinse them once with sterile water, put them in 0.6% sodium hypochlorite aqueous solution, shake them and soak them for 20 minutes, and ensure that the explants and Fully contact with disinfectant, rinse with sterile water 3 times, peel off the outermost layer of leaf sheath, smooth the incision position and remove the injured tissue after disinfection, and set aside.

In this embodiment, in the S02 step, the treated explants are inoculated into the adventitious bud induction medium for cultivation, and the adventitious bud seedlings can be obtained after culturing for 50 days. The cultivation environment temperature is 24°C, and the light duration is 10 hours. 800lux.

In the present embodiment, in the S03 step, the adventitious bud seedlings are divided into individual plants, the leaves are removed, and the stem section 1cm is reserved, and inoculated into the adventitious bud proliferation medium for cultivation. After 50 days of cultivation, the stem section re-grows 2- 3 seedlings with a multiplication coefficient of 2.0-3.0. Divide the grown adventitious bud seedlings into individual plants, cut off the leaves, keep 1 cm of the stem section, inoculate into the adventitious bud medium to continue the next generation culture, the total number of subculture multiplication is 15 times, and obtain robust The adventitious bud seedlings were cultivated at an ambient temperature of 24°C, with a light duration of 10 hours and a light intensity of 1500 lux.

In the present embodiment, in the S04 step, the robust adventitious buds are cut into individual seedlings, inoculated in the rooting medium and cultivated. After 30 days, a complete plant with 2-3 main roots can be obtained, and the rooting rate reaches 95%. %, the culture environment temperature is 24°C, the light duration is 10 hours, and the light intensity is 1500lux. .

In the present embodiment, in the S05 step, the complete plant seedlings are taken out from the culture bottle, the culture medium is washed off with clean water, transferred to a water curtain greenhouse, planted in a 72-hole tray, and drenched once 2000 times after planting Chlorothalonil, the planting environment humidity is 80%, the temperature is 26°C, and the planting substrate is peat: coconut peat = 4:1. The thickness of the peat is 5. After the first new leaf grows, it is poured with fertilizer and water, and the survival rate reaches 95%.

In this example, the media selected for the adventitious bud induction culture in the step S02, the proliferation culture for the adventitious buds in the step S03, and the rooting culture in the step S04 include: NH ₄ NO ₃ 990 mg/L, KNO ₃ 1140 mg/L L, MgSO ₄ 7H ₂ O2 22mg/L, KH ₂ PO ₄ 102mg/L, CaCl ₂ 2H ₂ O2 64mg/L, KI0.83mg/L, H ₃ BO ₃ 6.2mg/L, MnSO ₄ 4H ₂ O22. 3mg/L, ZnSO ₄ 7H ₂ O 8.6mg/L, Na ₂ MoO ₂ 2H ₂ O 0.25mg/L, CuSO ₄ 5H ₂ O 0.025mg/L, CoCl ₂ 6H ₂ O 0.025mg/L, FeSO ₄ 7H ₂ O 27.8mg/L, Na ₂ EDTA 37.3mg/L, Thiamine Hydrochloride 0.1mg/L, Niacin 0.5mg/L, Pyridoxine Hydrochloride 0.5mg/L, Glycine 2mg/L, Ascorbic acid 5mg/L, inositol 100mg/L.

In the present embodiment, in the S02 step, the induction medium is selected when explants are induced for adventitious buds, and the induction medium also includes 30 g/L of white sugar, 8.0 g/L of carrageenan, and 1.0 g/L of 6-benzylaminopurine. mg/L, Thidiazuron 0.01mg/L, Naphthaleneacetic acid 0.05mg/L.

In the present embodiment, in the S03 step, the proliferation medium is selected when the adventitious buds are proliferated, and the proliferation medium also includes 30 g/L of white sugar, 8.0 g/L of carrageenan, and 0.5 mg/L of 6-benzylaminopurine. L, thiadiuron 0.01mg/L, indole butyric acid 0.1mg/L.

In the present embodiment, in the S04 step, a rooting medium is used when a single seedling is rooted, and the rooting medium also includes 25 g/L of white sugar, 8.0 g/L of carrageenan, and 0.1 mg of 6-benzylaminopurine. /L, naphthaleneacetic acid 0.01mg/L, indole butyric acid 0.1mg/L, activated carbon 0.2g/L.

Example 2

A tissue culture rapid breeding method of green rainbow arrowroot, comprising:

S01: Before taking the explants from the female parent, perform irrigation treatment, cut the strong lateral buds from the female parent, soak, rinse and dry the incision, after ozone fumigation, then soak in hydrogen peroxide for 1.5 hours, rinse with sterile water, Shake and soak the explants in alcohol, rinse with sterile water, shake and soak in sodium hypochlorite aqueous solution, rinse with sterile water, smooth the incision and remove it for later use;

S02: Inducing and culturing the treated explants for adventitious buds until seedlings with adventitious buds are obtained;

S03: Divide the adventitious bud seedlings into individual plants, remove the leaves, keep the stem section, inoculate into the adventitious bud proliferation medium for cultivation, and subculture the new buds and original buds until multiple robust adventitious buds are obtained. The adventitious buds are multiplied;

S04: cut the vigorously propagated adventitious buds into individual seedlings, inoculate them in the rooting medium for cultivation, and obtain complete plant seedlings with taproots;

S05: Take out the complete plant seedlings from the culture bottle, wash off the medium, transfer to the water curtain greenhouse, and plant them in the 72-hole tray. After planting, they are sprayed with chlorothalonil, and after the first new leaf grows, they are sprayed with fertilizer and water , the survival rate was 95%.

In this embodiment, in the S01 step, before the explants are taken from the female parent, it is treated with 1500 times chlorothalonil irrigation, the temperature of the custody environment is kept at 24°C, the humidity is kept at 88%, and the light intensity is kept at 7500 lux. Wait for the peat humidity of the female parent to drop to 19%, cut off 3-5cm long healthy lateral buds from the female parent, peel off the old leaf sheaths, keep the innermost 2-3 layers of clean leaf sheaths, cut the incision flat with a scalpel, add water to wash Soak in detergent for 7.5 minutes, rinse with water and dry, fumigate with ozone for 50 minutes, soak in 5% hydrogen peroxide for 1.5 hours with shock, rinse with sterile water twice. Transfer the treated explants to the ultra-clean workbench, shake and soak them with 75% alcohol for 55 seconds, wash them once with sterile water, put them into 0.6% sodium hypochlorite aqueous solution, shake them and soak them for 25 minutes, and ensure that the explants are in good contact with the environment during disinfection. Fully contact with disinfectant, rinse with sterile water 3 times, peel off the outermost layer of leaf sheath, smooth the incision position and remove the injured tissue after disinfection, and set aside.

In this embodiment, in the S02 step, the treated explants are inoculated into the adventitious bud induction medium for cultivation, and the adventitious bud seedlings can be obtained after 55 days of cultivation. The cultivation environment temperature is 25° C., and the light duration is 10 hours. 1150lux.

In the present embodiment, in the S03 step, the adventitious bud seedlings are divided into individual plants, the leaves are removed, and the stem section 1cm is reserved, which is inoculated into the adventitious bud proliferation medium for cultivation. After 55 days of cultivation, the stem section re-grows 2- 3 seedlings with a multiplication coefficient of 2.0-3.0. Divide the grown adventitious bud seedlings into individual plants, cut off the leaves, keep the stem section 1-1.5 cm, inoculate into the adventitious bud medium to continue the next generation culture, and the total number of subcultures is 17 times To obtain robust adventitious bud seedlings, the culture environment temperature is 25°C, the light duration is 10 hours, and the light intensity is 2000lux.

In the present embodiment, in the S04 step, the vigorously propagated adventitious buds are cut into individual seedlings, inoculated in the rooting medium and cultivated. After 35 days, a complete plant with 2-3 main roots can be obtained, and the rooting rate reaches 96.5%. %, the culture environment temperature is 25°C, the light duration is 10 hours, and the light intensity is 2000lux.

In the present embodiment, in the S05 step, the complete plant seedlings are taken out from the culture bottle, the culture medium is washed off with clean water, transferred to a water curtain greenhouse, planted in a 72-hole tray, and drenched once 2000 times after planting Chlorothalonil, the planting environment humidity is 88%, the temperature is 28°C, the planting substrate is peat:coconut peat=4:1, the thickness of peat is 15, after the first new leaf grows, start to pour fertilizer and water, the survival rate up to 95%.

In this example, the media selected for the adventitious bud induction culture in the step S02, the proliferation culture for the adventitious buds in the step S03, and the rooting culture in the step S04 include: NH ₄ NO ₃ 990 mg/L, KNO ₃ 1140 mg/L L, MgSO ₄ 7H ₂ O2 22mg/L, KH ₂ PO ₄ 102mg/L, CaCl ₂ 2H ₂ O2 64mg/L, KI0.83mg/L, H ₃ BO ₃ 6.2mg/L, MnSO ₄ 4H ₂ O22. 3mg/L, ZnSO ₄ 7H ₂ O 8.6mg/L, Na ₂ MoO ₂ 2H ₂ O 0.25mg/L, CuSO ₄ 5H ₂ O 0.025mg/L, CoCl ₂ 6H ₂ O 0.025mg/L, FeSO ₄ 7H ₂ O 27.8mg/L, Na ₂ EDTA 37.3mg/L, Thiamine Hydrochloride 0.1mg/L, Niacin 0.5mg/L, Pyridoxine Hydrochloride 0.5mg/L, Glycine 2mg/L, Ascorbic acid 7.5mg/L, inositol 100mg/L.

In the present embodiment, in the S02 step, the induction medium is selected when explants are induced for adventitious buds, and the induction medium also includes white sugar 30g/L, carrageenan 8.0g/L, 6-benzylaminopurine 2.5 mg/L, Thidiazuron 0.045mg/L, Naphthaleneacetic acid 0.125mg/L.

In the present embodiment, in the S03 step, the proliferation medium is selected when the adventitious buds are proliferated, and the proliferation medium also includes 30 g/L of white sugar, 8.0 g/L of carrageenan, and 1.25 mg/L of 6-benzylaminopurine. L, thiadiuron 0.045mg/L, indole butyric acid 0.15mg/L.

In the present embodiment, in the S04 step, a rooting medium is used when a single seedling is rooted, and the rooting medium also includes 25 g/L of white sugar, 8.0 g/L of carrageenan, and 0.15 mg of 6-benzylaminopurine. /L, naphthaleneacetic acid 0.015mg/L, indolebutyric acid 0.25mg/L, activated carbon 0.2g/L.

Example 3

A tissue culture rapid breeding method of green rainbow arrowroot, comprising:

S01: Before taking the explants from the female parent, perform irrigation treatment, cut the healthy lateral buds from the female parent, soak, rinse and dry the incision, after ozone fumigation, then soak in hydrogen peroxide for 2 hours, rinse with sterile water, Shake and soak the explants in alcohol, rinse with sterile water, shake and soak in sodium hypochlorite aqueous solution, rinse with sterile water, smooth the incision and remove it for later use;

S02: Inducing and culturing the treated explants for adventitious buds until seedlings with adventitious buds are obtained;

S03: Divide the adventitious bud seedlings into individual plants, remove the leaves, keep the stem section, inoculate into the adventitious bud proliferation medium for cultivation, and subculture the new buds and original buds until multiple robust adventitious buds are obtained. The adventitious buds are multiplied;

S04: cut the vigorously propagated adventitious buds into individual seedlings, inoculate them in the rooting medium for cultivation, and obtain complete plant seedlings with taproots;

S05: Take out the complete plant seedlings from the culture bottle, wash off the medium, transfer to the water curtain greenhouse, and plant them in the 72-hole tray. After planting, they are sprayed with chlorothalonil, and after the first new leaf grows, they are sprayed with fertilizer and water , the survival rate was 95%.

In the present embodiment, in the step S01, the female parent is treated with 1500 times chlorothalonil before taking the explants, the temperature of the custody environment is kept at 28°C, the humidity is kept at 95%, and the light intensity is kept at 10000 lux. Wait for the peat humidity of the female parent to drop to 20%, cut off 3-5cm long healthy side buds from the female parent, peel off the old leaf sheaths, keep the innermost 2-3 layers of clean leaf sheaths, cut the incision flat with a scalpel, add water to wash Soak in detergent for 5-10 minutes, rinse with water and dry, fumigate with ozone for 40-60 minutes, soak in 5% hydrogen peroxide for 2 hours, rinse with sterile water twice. Transfer the treated explants to the ultra-clean workbench, shake and soak them with 75% alcohol for 70 seconds, wash them once with sterile water, put them in 0.6% sodium hypochlorite aqueous solution, shake them and soak them for 30 minutes, and ensure that the explants and Fully contact with disinfectant, rinse with sterile water 3 times, peel off the outermost layer of leaf sheath, smooth the incision position and remove the injured tissue after disinfection, and set aside.

In this embodiment, in the S02 step, the treated explants are inoculated into the adventitious bud induction medium for cultivation, and the adventitious bud seedlings can be obtained after 60 days of cultivation. The cultivation environment temperature is 26° C., and the light duration is 10 hours. 1500lux.

In the present embodiment, in the S03 step, the adventitious bud seedlings are divided into individual plants, the leaves are removed, and the stem section 1cm is reserved, which is inoculated into the adventitious bud proliferation medium for cultivation. After 60 days of cultivation, the stem section re-grows 2- 3 seedlings with a multiplication coefficient of 2.0-3.0. Divide the grown adventitious bud seedlings into individual plants, remove the leaves, keep the stem section 1-1.5 cm, inoculate into the adventitious bud medium to continue the next generation culture, and the total number of subcultures is 20 times To obtain robust adventitious bud seedlings, the culture environment temperature is 26°C, the light duration is 10 hours, and the light intensity is 2500lux.

In the present embodiment, in the S04 step, the vigorously propagated adventitious buds are cut into individual seedlings, inoculated in the rooting medium and cultivated. After 40 days, a complete plant with 2-3 main roots can be obtained, and the rooting rate reaches 98%. %, the culture environment temperature is 26°C, the light duration is 10 hours, and the light intensity is 2500lux. .

In the present embodiment, in the S05 step, the complete plant seedlings are taken out from the culture bottle, the culture medium is washed off with clean water, transferred to a water curtain greenhouse, planted in a 72-hole tray, and drenched once 2000 times after planting Chlorothalonil, the planting environment humidity is 95%, the temperature is 30°C, and the planting substrate is peat: coconut peat = 4:1. The thickness of the peat is 25. After the first new leaf grows, it is poured with fertilizer and water, and the survival rate reaches 95%.

In this example, the media selected for the adventitious bud induction culture in the step S02, the proliferation culture for the adventitious buds in the step S03, and the rooting culture in the step S04 include: NH ₄ NO ₃ 990 mg/L, KNO ₃ 1140 mg/L L, MgSO ₄ 7H ₂ O2 22mg/L, KH ₂ PO ₄ 102mg/L, CaCl ₂ 2H ₂ O2 64mg/L, KI0.83mg/L, H ₃ BO ₃ 6.2mg/L, MnSO ₄ 4H ₂ O22. 3mg/L, ZnSO ₄ 7H ₂ O 8.6mg/L, Na ₂ MoO ₂ 2H ₂ O 0.25mg/L, CuSO ₄ 5H ₂ O 0.025mg/L, CoCl ₂ 6H ₂ O 0.025mg/L, FeSO ₄ 7H ₂ O 27.8mg/L, Na ₂ EDTA 37.3mg/L, Thiamine Hydrochloride 0.1mg/L, Niacin 0.5mg/L, Pyridoxine Hydrochloride 0.5mg/L, Glycine 2mg/L, Ascorbic acid 10mg/L, inositol 100mg/L.

In this embodiment, in the S02 step, the induction medium is selected when explants are subjected to adventitious bud induction culture, and the induction medium also includes 30 g/L of white sugar, 8.0 g/L of carrageenan, and 4.0 g/L of 6-benzylaminopurine. mg/L, Thidiazuron 0.08mg/L, Naphthaleneacetic acid 0.2mg/L.

In the present embodiment, in the S03 step, the proliferation medium is selected when the adventitious buds are proliferated, and the proliferation medium also includes 30 g/L of white sugar, 8.0 g/L of carrageenan, and 2.0 mg/L of 6-benzylaminopurine. L, thiadiuron 0.08mg/L, indole butyric acid 0.2mg/L.

In the present embodiment, in the S04 step, a rooting medium is used when a single seedling is rooted, and the rooting medium also includes 25 g/L of white sugar, 8.0 g/L of carrageenan, and 0.2 mg of 6-benzylaminopurine. /L, naphthaleneacetic acid 0.02mg/L, indole butyric acid 0.4mg/L, activated carbon 0.2g/L.

In addition, the inventors of the present invention found that the addition of low concentration of thiadizuron in the adventitious bud induction culture and the proliferation medium formula of adventitious buds helps to promote the bud differentiation in the proliferation process, and at the same time promote the bud expansion, which improves the proliferation coefficient and bud growth rate. The survival rate of adventitious buds is increased by adding ascorbic acid to the culture medium of adventitious bud induction, proliferation of adventitious buds, and rooting culture to inhibit the accumulation of quinones in the medium, reduce the toxicity of quinones to the proliferation of buds, and improve the absorption of value-added buds Nutritional efficiency and improve the survival rate of rooted seedlings. Adding low concentration of 6-benzylaminopurine to the rooting medium can make the plants grow robustly, increase the chlorophyll content of leaves, and brighten the leaves. The ambient temperature is 24-26°C and the light duration is 10 hours. , the light intensity is 1500-2500lux, all of which can reproduce robust tissue culture seedlings stably for a long time, the rooting rate is over 95%, and the root system is healthy. In the transplanting step of tissue culture seedlings, the substrate for planting is peat:coconut peat=4:1 , greatly improving the survival rate of tissue culture seedlings after planting, and can make the survival rate of tissue culture seedlings after planting reach 95%.

It will be apparent to those skilled in the art that the invention is not limited to the details of the above-described exemplary embodiments, but that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Accordingly, the embodiments should be regarded in all points of view as exemplary and not restrictive, the scope of the invention being defined by the appended claims rather than the foregoing description, and it is therefore intended that the scope of the invention be defined by the appended claims rather than by the foregoing description. All changes within the meaning and range of equivalents of the elements are embraced in the present invention. Any reference sign in a claim should not be construed as limiting the claim concerned.

Claims (10)

1. A tissue culture rapid breeding method of green rainbow Maranta arundinacea is characterized by comprising the following steps:

s01: performing perfusion treatment before the explant is taken from the female parent, cutting robust lateral buds from the female parent, soaking, washing and drying the cut, after ozone fumigation, performing vibration soaking for 1-2 hours by using hydrogen peroxide, washing with sterile water, performing dragging soaking on the explant by using alcohol, washing with the sterile water, performing dragging soaking in a sodium hypochlorite aqueous solution, washing with the sterile water, flattening the cut, and cutting for later use;

s02: carrying out adventitious bud induction culture on the treated explant until adventitious bud plantlets are obtained;

s03: dividing adventitious bud plantlets into single plants, cutting off leaves, reserving stem segments, inoculating the single plants into an adventitious bud multiplication culture medium for culture, carrying out subculture multiplication on new buds and original buds until a plurality of strong adventitious buds are obtained, and carrying out propagation on the induced adventitious buds;

s04: cutting the strong adventitious bud into single plantlet, inoculating in rooting culture medium, and culturing to obtain complete plantlet with main root;

s05: taking out the whole plant plantlet from the culture bottle, washing the culture medium, transferring to a water curtain greenhouse, planting in a 72-hole tray, spraying chlorothalonil after planting, and spraying rich water after growing a first new leaf, wherein the survival rate reaches 95%.

2. The tissue culture rapid propagation method of the green rainbow Maranta arundinacea according to claim 1, characterized in that in the step S01, before the female parent takes the explant, the explant is treated by 1500 times of chlorothalonil by drenching, the temperature of the culture environment is kept at 20-28 ℃, the humidity is kept at 80-95%, the illumination intensity is kept at 5000-10000lux,

waiting for the humidity of the peat of the female parent to be reduced to 18-20%, cutting 3-5cm long strong lateral buds from the female parent, stripping old leaf sheaths, keeping 2-3 clean leaf sheaths at the innermost layer, cutting the cut smoothly by using a scalpel, adding detergent into clear water, soaking for 5-10 minutes, washing with clear water, drying in the air, fumigating with ozone for 40-60 minutes, shaking and soaking with 5% hydrogen peroxide for 1-2 hours, and washing with sterile water for 2 times; transferring the processed explant to a super-clean workbench, shaking and soaking the explant for 40-70s by using 75% alcohol, washing the explant by using sterile water for 1 time, putting the explant into 0.6% sodium hypochlorite aqueous solution, shaking and soaking the explant for 20-30 minutes, ensuring that the explant is fully contacted with disinfectant during disinfection, washing the explant by using the sterile water for 3 times, stripping an outermost layer of leaf sheath, flattening the incision position, and cutting the disinfected injured tissue for later use.

3. The tissue culture rapid propagation method of green rainbow Maranta arundinacea according to claim 1, characterized in that in the step S02, the processed explant is inoculated into an adventitious bud induction culture medium for culture, adventitious bud plantlets can be obtained after 50-60 days of culture, the culture environment temperature is 24-26 ℃, the illumination time is 10 hours, and the illumination intensity is 800-1500lux.

4. The tissue culture rapid propagation method of the green rainbow Maranta arundinacea according to claim 1, characterized in that in the step S03, adventitious bud plantlets are divided into single plants, leaves are cut off, stem sections are kept for 1cm, the individual plants are inoculated into an adventitious bud multiplication culture medium for culture, after 50-60 days of culture, 2-3 plantlets are grown from the stem sections again, the multiplication coefficient is 2.0-3.0, the grown adventitious bud plantlets are divided into single plants, leaves are cut off, stem sections are kept for 1-1.5cm, the individual plants are inoculated into the adventitious bud culture medium for continuous next generation culture, the total number of subculture multiplication is 15-20 times, strong adventitious bud plantlets are obtained, the culture environment temperature is 24-26 ℃, the illumination time is 10 hours, and the illumination intensity is 1500-lux.

5. The tissue culture rapid propagation method of the green rainbow Maranta arundinacea as claimed in claim 1, wherein in the step S04, the strongly propagated adventitious buds are cut into single seedlings, the single seedlings are inoculated in a rooting culture medium for culture, 2-3 complete plants with main roots can be obtained after 30-40 days, the rooting rate reaches 95-98%, the culture environment temperature is 24-26 ℃, the illumination time is 10 hours, and the illumination intensity is 1500-2500lux.

6. The tissue culture rapid propagation method of the green rainbow arrowroot as claimed in claim 1, wherein in the step S05, whole plant plantlets are taken out of a culture bottle, the culture medium is washed away by clean water, the plantlets are transferred to a water curtain greenhouse and planted in a 72-hole tray, 2000 times of chlorothalonil is sprayed once after planting, the humidity of the planting environment is 80-95%, the temperature is 26-30 ℃, and the planted substrate is peat: the coconut husk =4,

the thickness of peat is 5-25, after the peat is equilong to obtain the first new leaf, the fertilizer water is sprayed, and the survival rate is up to 95%.

7. The tissue culture and rapid propagation method of Maranta marmorata as claimed in claim 1, wherein the step S02 comprises adventitious bud induction culture, the step S03 comprises adventitious bud proliferation culture, and the step S04 comprisesThe culture medium selected for rooting culture of the rows comprises: NH ₄ NO ₃ 990mg/L、KNO ₃ 1140mg/L、MgSO ₄ ·7H ₂ O222mg/L、KH ₂ PO ₄ 102mg/L、CaCl ₂ ·2H ₂ O264mg/L、KI0.83mg/L、H ₃ BO ₃ 6.2mg/L、MnSO ₄ ·4H ₂ O22.3mg/L、ZnSO ₄ ·7H ₂ O8.6mg/L、Na ₂ MoO ₂ ·2H ₂ O 0.25mg/L、CuSO ₄ ·5H ₂ O 0.025mg/L、CoCl ₂ ·6H ₂ O 0.025mg/L、FeSO ₄ ·7H ₂ O 27.8mg/L、Na ₂ 37.3mg/L of EDTA, 0.1mg/L of thiamine hydrochloride, 0.5mg/L of nicotinic acid, 0.5mg/L of pyridoxine hydrochloride, 2mg/L of glycine, 5-10mg/L of ascorbic acid, and 100mg/L of inositol.

8. The tissue culture rapid propagation method of the green rainbow Maranta arundinacea according to claim 1, characterized in that in the step S02, an induction culture medium is selected when adventitious bud induction culture is performed on an explant, and the induction culture medium further comprises 30g/L of white sugar, 8.0g/L of carrageenan, 1.0-4.0mg/L of 6-benzylaminopurine, 0.01-0.08mg/L of thidiazuron and 0.05-0.2mg/L of naphthylacetic acid.

9. The tissue culture rapid propagation method of arum polygama according to claim 1, wherein in the step S03, a propagation culture medium is selected for propagation culture of adventitious buds, and the propagation culture medium further comprises 30g/L of white sugar, 8.0g/L of carrageenan, 0.5-2.0mg/L of 6-benzylaminopurine, 0.01-0.08mg/L of thidiazuron, and 0.1-0.2mg/L of indolebutyric acid.

10. The preparation method of tissue culture rapid propagation of Maranta arundinacea according to claim 1, wherein in the step S04, a rooting culture medium is adopted for rooting culture of the single plantlet, and the rooting culture medium further comprises 25g/L of white sugar, 8.0g/L of carrageenan, 0.1-0.2mg/L of 6-benzylaminopurine, 0.01-0.02mg/L of naphthylacetic acid, 0.1-0.4mg/L of indolebutyric acid, and 0.2g/L of activated carbon.

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