Tissue culture and rapid propagation method for arrowroot

Abstract

The invention discloses a tissue culture and rapid propagation method for arrowroot. The tissue culture and rapid propagation method comprises the following steps: 1) obtaining and disinfecting an explant; 2) adventitious bud induction; (3) carrying out subculture multiplication; 4) rooting and seedling strengthening; and 5) transplanting test-tube plantlets. According to the method, explant adventitious bud induction, multiplication culture and rooting culture are carried out by selecting a new bud of arrowroot as an explant, optimizing a disinfection step and selecting an improved MS culture medium and an improved 1 / 2 MS culture medium to replace a common MS culture medium; the disinfection survival rate of arrowroot, the induction rate of adventitious buds, the multiplication coefficient of cluster buds, the rooting rate of seedlings and the transplanting survival rate are remarkably improved, and a large number of test-tube plantlets can be bred in a short time to meet the market requirements. The technology is simple, affordable, feasible and high in application value. The method can be implemented only by simple plant tissue culture equipment.

Description

A rapid propagation method for Calathea sylvestris tissue culture Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically relating to a method for rapid propagation of Maranta variegata through tissue culture. Background Technology

[0002] Calathea spp., belonging to the family Marantaceae, is a perennial evergreen herbaceous plant native to Brazil. Its leaves are long and lanceolate, green on the upper surface with prominent wavy folds along the midrib, and purplish-red on the underside. This shade-tolerant plant is suitable for indoor decoration and landscaping, making it an excellent indoor foliage plant and an important ornamental plant for gardens and urban beautification. Traditional methods of propagating Calathea spp. typically include division and cuttings, but these methods have low propagation coefficients and result in a very limited number of seedlings. This invention patent utilizes the base of young shoots of Calathea spp. as explants for tissue culture, enabling the rapid propagation of a large number of test-tube seedlings to meet market demand. Summary of the Invention

[0003] The purpose of this invention is to address the aforementioned shortcomings by providing a rapid propagation method for Calathea elegans through tissue culture. This invention is simple, affordable, practical, and has high application value; its implementation requires only simple plant tissue culture equipment.

[0004] The technical solution adopted in this invention is as follows:

[0005] A rapid propagation method for Calathea variegata tissue culture, characterized by comprising the following steps:

[0006] a. Disinfection and induction culture of explants: Select disease-free *Calathea variegata* parent plants and take newly grown trumpet-shaped shoots as explants. After digging the shoots from the ground, do not wash them. First, cut off the upper leaves and remove the outer bracts. Then, disinfect the 3-4 cm stem base and inoculate them in the induction medium until adventitious shoots of the explants grow. The composition of the induction medium is: modified MS medium + 4.0-6.0 mg / L 6-BA + 0.3-0.8 mg / L KT + 0.1-0.2 mg / L NAA + 20-25 g / L sucrose + 6-6.5 g / L agar, pH 5.7-6.0;

[0007] b. Subculture and proliferation: Remove the leaves and stems from the obtained adventitious buds, leaving the bud base, and inoculate it onto the proliferation medium to form clustered buds; the subculture and proliferation medium is: modified MS medium + 2.0-4.0 mg / L 6-BA + 0.1-0.5 mg / L KT + 0.1-0.3 mg / L NAA + 20-30 g / L sucrose + 5-7 g / L agar, pH 5.8-6.0;

[0008] c. Rooting and seedling cultivation: When the shoot clusters are 2-3 cm tall, cut off the obtained shoots and inoculate them onto a rooting medium for rooting. The rooting medium is: modified 1 / 2 MS medium + 0.3-1.0 mg / L NAA + 20-30 g / L sucrose + 6-7 g / L agar, pH 5.8-6.0.

[0009] d. Transplanting of test-tube seedlings: When the rooted test-tube seedlings grow to 4-5cm, transfer them to natural light for 3-10 days to harden them off. Then, open the bottle stopper, use tweezers to remove the test-tube seedlings from the culture bottle, wash off the culture medium from the roots, and plant them in the substrate for culture.

[0010] The improved MS medium is prepared by adjusting the concentration of ammonium nitrate to 1900 mg / L, potassium nitrate to 1500 mg / L, and magnesium sulfate to 450 mg / L, while keeping the other components unchanged.

[0011] The modified 1 / 2MS medium is prepared by halving the amount of macroelements in the MS medium formula, adjusting the concentration of the organic components thioammonium hydrochloride to 2.0 mg / L, pyridoxine hydrochloride to 1.0 mg / L, and nicotinic acid to 1.0 mg / L, while keeping the other components unchanged.

[0012] Preferably, the explants mentioned in step a are obtained through the following pretreatment method: Select new shoots of mature Maranta variegata as materials. 42 days before the explants are selected and disinfected, irrigate once with a 500-800 times diluted solution of 50% carbendazim wettable powder and spray the leaves. 7 days later, irrigate once with a 3500-4500 times diluted solution of agricultural thiophanate-methyl·fludioxonil soluble liquid and spray the leaves. Repeat the above steps for another 7 days, for a total of 4 treatments. 7 days after the last irrigation with the thiophanate-methyl·fludioxonil aqueous solution, select the treated new shoots of Maranta variegata as explants.

[0013] Preferably, the disinfection of the stem base in step a involves wiping the surface of the explant with a cotton ball soaked in 75% ethanol. After pretreatment, the explant is soaked in 75% ethanol for 20-60 seconds, and then disinfected several times with 0.1% mercuric chloride solution. After each disinfection, the explant is rinsed with sterile water 2-6 times.

[0014] Preferably, the method of continuously disinfecting with 0.1% mercuric chloride solution for several times specifically means: continuously disinfecting with 0.1% mercuric chloride solution for 3 times, with the first disinfection time being 4-7 minutes, the second disinfection time being 3-6 minutes, and the third disinfection time being 1-4 minutes.

[0015] Preferably, the cultivation conditions for step a are: cultivation temperature 22-27℃, light intensity 500-1000lx, and light intensity 9-13 h / d.

[0016] Preferably, the culture conditions for steps b and c are: culture temperature 22-27℃, light intensity 1000-2000lx, and light intensity 9-13h / d.

[0017] Preferably, the matrix in step d is a mixed matrix of peat soil and perlite in a volume ratio of (2-4):(1-3).

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. Feasibility of Maranta regeneration: The culture medium of the present invention provides a way to induce Maranta regeneration under tissue culture conditions, which allows a large number of genetically consistent offspring to be rapidly propagated from a single parent plant, increasing the feasibility of Maranta regeneration.

[0020] 2. Growth control: By adjusting the concentration of plant growth hormones and growth-promoting factors in the culture medium, precise control of the growth of arrowroot tissues can be achieved, which helps to optimize the regeneration process.

[0021] 3. In summary, this invention provides an effective tool for the in vitro regeneration of arrowroot, which helps to accelerate the propagation and growth process of arrowroot, improve the controllability of growth conditions, reduce costs, and contribute to the sustainable cultivation of arrowroot. Attached Figure Description

[0022] Figure 1 shows the in vitro rapid propagation system of Calathea pulidonis. A, B: adventitious bud induction culture; C, D: proliferation culture and differentiation into seedlings; E, F: rooting induction. Detailed Implementation

[0023] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0024] Example 1:

[0025] 1. Explant sterilization and adventitious bud induction: The explants of *Calathea spinulosa* carry dust and microorganisms on the outside and contain endophytic bacteria inside. Thorough sterilization is necessary without damaging plant cells; therefore, successful explant sterilization is the first step in tissue culture. During the budding season, select vigorous, disease-free, and healthy *Calathea spinulosa* strains grown in greenhouses. 42 days before inoculation, irrigate and spray the leaves once with a 600-fold dilution of 50% carbendazim wettable powder. Seven days later, irrigate and spray the leaves once with a 4000-fold dilution of agricultural chlorothalonil-fludioxonil soluble liquid (manufacturer: Syngenta Nantong Crop Protection Co., Ltd.). Repeat the above steps for another seven days, for a total of four treatments. Seven days after the final irrigation with the chlorothalonil-fludioxonil solution, select the treated *Calathea spinulosa* shoots as explants for tissue culture.

[0026] Newly sprouted trumpet-shaped shoots were selected as explants. After being dug up from the ground, the shoots were not washed. The upper leaves and outer bracts were removed. The 3-4 cm stem base was then disinfected. The explant surface was first wiped with cotton balls soaked in a 75% ethanol solution, then immersed in the 75% ethanol solution for 20 seconds, disinfected with a 0.1% mercuric chloride solution for 5 minutes, rinsed twice with sterile water, disinfected with a 0.1% mercuric chloride solution for 4 minutes, rinsed twice with sterile water, and disinfected with a 0.1% mercuric chloride solution for 2 minutes, rinsed twice with sterile water. The disinfected explants were then inoculated onto adventitious bud induction medium for induction. The culture temperature was 24℃, the light intensity was 500 lx, and the light duration was 9 h / d. Adventitious buds formed after 20 days of culture, with a disinfection success rate of 78%. The induction medium is: the basal medium is MS medium (control) or a modified MS medium (the concentration of ammonium nitrate in the ordinary MS medium formula is adjusted to 1900 mg / L, the concentration of potassium nitrate is adjusted to 1500 mg / L, and the concentration of magnesium sulfate is adjusted to 450 mg / L, while the other components remain unchanged) + 4.0 mg / L 6-BA + 0.8 mg / L KT + 0.1 mg / L NAA + 20 g / L sucrose + 6.0 g / L agar, pH 5.7. The preparation method is to add each component to the basal medium, adjust the pH value, and sterilize for later use.

[0027] Experimental Results: Different basal culture media had significant effects on the induction of adventitious shoots in *Calathea variegata*. With MS as the basal medium, the induction rate of explants was 80.60%, while with the modified MS as the basal medium, the induction rate reached 86.80% (20 days post-inoculation), which was 6.20% higher than that with MS as the basal medium. Furthermore, shoot germination was observed 5-7 days post-inoculation, 2-3 days earlier than with MS as the basal medium. Therefore, the modified MS medium was selected as the most suitable basal medium for explant induction.

[0028] 2. Subculture and Proliferation: Leaves and stems were removed from the induced adventitious buds, and the basal parts of the buds were inoculated onto the proliferation medium for culture to form clustered buds. The culture temperature was 24℃, the light intensity was 1000 lx, and the photoperiod was 13 h / d. One subculture and proliferation cycle was 45 days. After four consecutive culture cycles, the fold increase after each subculture and proliferation cycle was 4.0 times. The proliferation medium was: a modified MS medium as the basal medium (the concentrations of ammonium nitrate, potassium nitrate, and magnesium sulfate in the ordinary MS medium formula were adjusted to 1900 mg / L, 1500 mg / L, and 450 mg / L, while the other components remained unchanged) + 2.0 mg / L 6-BA + 0.3 mg / L KT + 0.1 mg / L NAA + 20 g / L sucrose + 6.0 g / L agar, pH 5.9. The preparation method was to add each component to the basal medium, adjust the pH, and sterilize for later use.

[0029] 3. Rooting Culture: When the shoots reach a height of 3 cm, the obtained adventitious shoots are cut off and inoculated onto rooting medium for rooting. The culture is carried out at a temperature of 24-25℃, a light intensity of 1000 lx, and a light intensity of 13 h / d to obtain rooted tissue culture seedlings. After 30 days of culture, the rooting rate reaches 92%, with each seedling having 5 roots. The rooting medium is: a modified 1 / 2 MS medium as the basal medium (the amount of macroelements in the ordinary MS medium formula is halved, and the concentrations of the organic components, such as thioammonium hydrochloride, pyridoxine hydrochloride, and nicotinic acid, are adjusted to 2.0 mg / L, 1.0 mg / L, and 1.0 mg / L, while the other components remain unchanged) + 0.3 mg / L NAA + 20 g / L sucrose + 6.0 g / L agar, pH 5.8. The preparation method involves adding each component to the basal medium, adjusting the pH, and sterilizing for later use.

[0030] 4. Transplanting of test-tube seedlings: When the rooted test-tube seedlings grow to 5cm in height, transfer them to natural light for 3 days to harden them off. Then, open the bottle stopper, use tweezers to remove the test-tube seedlings from the culture bottle, wash off the culture medium from the roots, and plant them in a substrate with a peat moss:perlite ratio of 3:1 by volume. Pay attention to watering, shading, keeping warm and moist. The survival rate can reach 95% (Figure 1).

[0031] Example 2:

[0032] 1. Explant sterilization and adventitious bud induction: The explants of *Calathea spinulosa* carry dust and microorganisms on the outside and contain endophytic bacteria inside. Thorough sterilization is necessary without damaging plant cells; therefore, successful explant sterilization is the first step in tissue culture. During the budding season, select vigorous, disease-free, and healthy strains from greenhouses. 42 days before inoculation, irrigate and spray the leaves once with a 600-fold dilution of 50% carbendazim wettable powder. Seven days later, irrigate and spray the leaves once with a 4000-fold dilution of agricultural fungicide-fludioxonil soluble liquid. Repeat this process for another seven days, for a total of four treatments. Seven days after the final irrigation with the fungicide-fludioxonil solution, select the treated *Calathea spinulosa* shoots as explants for tissue culture.

[0033] Newly sprouted trumpet-shaped shoots of *Calathea spinulosa* were selected as explants. After being dug up from the ground, the shoots were not washed. The upper leaves and outer bracts were removed. The 3-4 cm stem base was then disinfected. The explant surface was first wiped with cotton balls soaked in a 75% ethanol solution, then immersed in the 75% ethanol solution for 30 seconds, disinfected with a 0.1% mercuric chloride solution for 6 minutes, rinsed three times with sterile water, disinfected with a 0.1% mercuric chloride solution for 5 minutes, rinsed three times with sterile water, and then disinfected with a 0.1% mercuric chloride solution for 3 minutes, rinsed three times with sterile water. The disinfected explants were then inoculated onto adventitious bud induction medium for induction. The culture temperature was 24℃, the light intensity was 700 lx, and the photoperiod was 11 h / d. Adventitious buds formed after 15 days of culture, with an adventitious bud occurrence rate of 90% (explants) (20 days post-inoculation), and a disinfection success rate of 82%. The induction medium consists of: a modified MS medium (the concentrations of ammonium nitrate, potassium nitrate, and magnesium sulfate in the ordinary MS medium formula are adjusted to 1900 mg / L, 1500 mg / L, and 450 mg / L, while the other components remain unchanged) + 5.0 mg / L 6-BA + 0.5 mg / L KT + 0.15 mg / L NAA + 25 g / L sucrose + 6.5 g / L agar, pH 5.8. The preparation method involves adding each component to the basal medium, adjusting the pH, and sterilizing for later use.

[0034] 2. Subculture and Proliferation: Leaves and stems were removed from the induced adventitious buds, leaving the bud base, which was then inoculated onto a proliferation medium for culture to form clustered buds. The culture temperature was 24℃, the light intensity was 1500 lx, and the photoperiod was 11 h / d. One subculture cycle was 45 days. After four consecutive subculture cycles, the fold increase after each subculture cycle was 4.5 times. The proliferation medium consisted of: a modified MS medium (the concentrations of ammonium nitrate, potassium nitrate, and magnesium sulfate in the ordinary MS medium formula were adjusted to 1900 mg / L, 1500 mg / L, and 450 mg / L, while the other components remained unchanged) + 3.0 mg / L 6-BA + 0.3 mg / L KT + 0.2 mg / L NAA + 25 g / L sucrose + 6.5 g / L agar, pH 5.8. The preparation method involved adding each component to the basal medium, adjusting the pH, and sterilizing for later use.

[0035] 3. Rooting Culture: When the shoots reach a height of 3 cm, the obtained adventitious shoots are cut off and inoculated onto rooting medium for rooting. The culture is carried out at a temperature of 24-25℃, a light intensity of 1500 lx, and a light intensity of 11 h / d to obtain rooted tissue culture seedlings. After 30 days of culture, the rooting rate can reach 96%, with each seedling having 5-6 roots. The rooting medium is: a modified 1 / 2 MS medium (the amount of macroelements in the ordinary MS medium formula is halved, and the concentrations of the organic components, such as thioammonium hydrochloride, pyridoxine hydrochloride, and nicotinic acid, are adjusted to 2.0 mg / L, 1.0 mg / L, and 1.0 mg / L, while the other components remain unchanged) + 0.6 mg / L NAA + 20 g / L sucrose + 6.0 g / L agar, pH 5.8. The preparation method involves adding each component to the basal medium, adjusting the pH, and sterilizing for later use.

[0036] 4. Transplanting of test-tube seedlings: When the rooted test-tube seedlings grow to 5cm in height, move them to natural light and harden them off for 5 days. Then, open the bottle stopper, use tweezers to remove the test-tube seedlings from the culture bottle, wash off the culture medium from the roots, and plant them in a substrate with a peat moss:perlite ratio of 4:2 by volume. Pay attention to watering, shading, keeping warm and moist. The survival rate can reach 96%.

[0037] Example 3:

[0038] 1. Explant sterilization and adventitious bud induction: The explants of *Calathea spinulosa* carry dust and microorganisms on the outside and contain endophytic bacteria inside. Thorough sterilization is necessary without damaging plant cells; therefore, successful explant sterilization is the first step in tissue culture. During the budding season, select vigorous, disease-free, and healthy strains from greenhouses. 42 days before inoculation, irrigate and spray the leaves once with a 600-fold dilution of 50% carbendazim wettable powder. Seven days later, irrigate and spray the leaves once with a 4000-fold dilution of agricultural fungicide-fludioxonil soluble liquid. Repeat this process for another seven days, for a total of four treatments. Seven days after the final irrigation with the fungicide-fludioxonil solution, select the treated *Calathea spinulosa* shoots as explants for tissue culture.

[0039] Newly sprouted trumpet-shaped shoots of *Calathea spinulosa* were selected as explants. After the shoots were dug up from the ground, they were not washed. The upper leaves were cut off and the outer bracts were removed. The 3-4 cm stem base was then disinfected. The surface of the explants was first wiped with cotton balls soaked in a 75% ethanol aqueous solution, then soaked in a 75% ethanol aqueous solution for 40 seconds, disinfected with a 0.1% mercuric chloride solution for 7 minutes, rinsed 4 times with sterile water, disinfected with a 0.1% mercuric chloride solution for 6 minutes, rinsed 4 times with sterile water, disinfected with a 0.1% mercuric chloride solution for 4 minutes, and rinsed 4 times with sterile water. The disinfected explants were then inoculated onto adventitious shoot induction medium for adventitious shoot induction. The culture temperature was 24℃, the light intensity was 1000 lx, and the light intensity was 13 h / d. Adventitious buds formed after 15 days of culture, with an adventitious bud occurrence rate (explant induction rate) of 85% (20 days after inoculation). The sterilization success rate was 80%, and some plants showed browning. The induction medium consisted of: modified MS medium (the concentrations of ammonium nitrate, potassium nitrate, and magnesium sulfate in the ordinary MS medium formula were adjusted to 1900 mg / L, 1500 mg / L, and 450 mg / L, while the other components remained unchanged) + 6.0 mg / L 6-BA + 0.3 mg / L KT + 0.2 mg / L NAA + 25 g / L sucrose + 6.5 g / L agar, pH 5.8. The preparation method involved adding each component to the basal medium, adjusting the pH, and sterilizing for later use.

[0040] 2. Subculture and Proliferation: Leaves and stems were removed from the induced adventitious buds, and the basal parts of the buds were inoculated onto the proliferation medium for culture to form clustered buds. The culture temperature was 24℃, the light intensity was 2000 lx, and the photoperiod was 9 h / d. One subculture cycle was 45 days. After four consecutive subculture cycles, the fold increase after each subculture cycle was 4.2 times. The proliferation medium was: a modified MS medium (the concentrations of ammonium nitrate, potassium nitrate, and magnesium sulfate in the ordinary MS medium formula were adjusted to 1900 mg / L, 1500 mg / L, and 450 mg / L, while the other components remained unchanged) + 4.0 mg / L 6-BA + 0.3 mg / L KT + 0.3 mg / L NAA + 30 g / L sucrose + 7.0 g / L agar, pH 5.9. The preparation method involved adding each component to the basal medium, adjusting the pH, and sterilizing for later use.

[0041] 3. Rooting Culture: When the shoots reach a height of 3 cm, the obtained adventitious shoots are cut off and inoculated onto rooting medium for rooting. The culture is carried out at a temperature of 24-25℃, a light intensity of 2000 lx, and a light intensity of 9 h / d. Rooted tissue culture seedlings are obtained. After 30 days of culture, the rooting rate can reach 95%, with each seedling having 5-6 roots. The rooting medium is: a modified 1 / 2 MS medium (the amount of macroelements in the ordinary MS medium formula is halved, and the concentrations of the organic components—thioammonium hydrochloride, pyridoxine hydrochloride, and nicotinic acid—are adjusted to 2.0 mg / L, 1.0 mg / L, and 1.0 mg / L, respectively, while the other components remain unchanged) + 1.0 mg / L NAA + 30 g / L sucrose + 7.0 g / L agar, pH 5.9. The preparation method involves adding each component to the basal medium, adjusting the pH, and sterilizing for later use.

[0042] 4. Transplanting of test-tube seedlings: When the rooted test-tube seedlings grow to 5cm in height, move them to natural light for 8 days to harden them off. Then, open the bottle stopper, use tweezers to remove the test-tube seedlings from the culture bottle, wash off the culture medium from the roots, and plant them in a substrate with a peat moss:perlite ratio of 4:1 by volume. Pay attention to watering, shading, keeping warm and moist. The survival rate can reach 92%.

[0043] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for rapid propagation of Maranta variegata through tissue culture, characterized in that, Includes the following steps: a. Disinfection and induction culture of explants: Select disease-free *Calathea variegata* parent plants and take newly grown trumpet-shaped shoots as explants. After digging the shoots from the ground, do not wash them. First, cut off the upper leaves and remove the outer bracts. Then, disinfect the 3-4 cm stem base and inoculate them into an induction medium until adventitious shoots emerge. The induction medium consists of: modified MS medium + 4.0-6.0 mg / L 6-BA + 0.3-0.8 mg / L KT + 0.1-0.2 mg / L NAA + 20-25 g / L sucrose + 6-6.5 g / L agar, pH 5.7-6.0; b. Subculture and proliferation culture: Cut off the leaves and stems of the obtained adventitious shoots, leaving the shoot base, and inoculate them into a proliferation medium to form clustered shoots. The subculture and proliferation medium consists of: modified MS medium + 2.0-4.0 mg / L 6-BA + 0.1-0.5 mg / L KT + 0.1-0.3 mg / L NAA + 20-30 g / L sucrose + 5-7 g / L agar, pH 5.8-6.0; c. Rooting and seedling cultivation: When the shoot clusters are 2-3 cm tall, the obtained shoots are cut off and inoculated onto rooting medium for rooting; the rooting medium is: modified 1 / 2 MS medium + 0.3-1.0 mg / L NAA + 20-30 g / L sucrose + 6-7 g / L agar, pH 5.8-6.

0. 5.8-6.0; d. Transplanting of test-tube seedlings: When the rooted test-tube seedlings grow to 4-5cm, transfer them to natural light for 3-10 days to harden them off. Then, open the bottle stopper, use tweezers to remove the seedlings from the culture bottle, wash off the culture medium from the roots, and plant them in the substrate for culture. The modified MS medium is prepared by adjusting the concentration of ammonium nitrate to 1900mg / L, potassium nitrate to 1500mg / L, and magnesium sulfate to 450mg / L, while keeping the other components unchanged. The modified 1 / 2 MS medium is prepared by halving the amount of macroelements in the MS medium formula, adjusting the concentration of the organic components, such as thioammonium hydrochloride, pyridoxine hydrochloride, and nicotinic acid, to 2.0mg / L, 1.0mg / L, and 1.0mg / L, while keeping the other components unchanged.

2. The method according to claim 1, characterized in that, The explants mentioned in step a were obtained through the following pretreatment method: Select new shoots of mature Maranta variegata as materials. 42 days before the explants were selected and disinfected, irrigate them once with a 500-800 times diluted solution of 50% carbendazim wettable powder and spray the leaves. 7 days later, irrigate them once with a 3500-4500 times diluted solution of agricultural cyprodinil·fludioxonil soluble liquid and spray the leaves. Repeat the above steps for another 7 days, for a total of 4 treatments. 7 days after the last irrigation with the cyprodinil·fludioxonil aqueous solution, select the treated new shoots of Maranta variegata as explants.

3. The method according to claim 1, characterized in that, The disinfection of the stem base mentioned in step a involves wiping the surface of the explant with a cotton ball soaked in 75% ethanol. After the pretreatment, the explant is soaked in 75% ethanol for 20-60 seconds, and then disinfected several times with 0.1% mercuric chloride solution. After each disinfection, the explant is rinsed with sterile water 2-6 times.

4. The method according to claim 3, characterized in that, The aforementioned disinfection with 0.1% mercuric chloride solution for several consecutive times specifically refers to disinfecting three times consecutively with 0.1% mercuric chloride solution. The first disinfection time is 4-7 minutes, the second disinfection time is 3-6 minutes, and the third disinfection time is 1-4 minutes.

5. The method according to claim 1, characterized in that, The cultivation conditions for step a are: cultivation temperature 22-27℃, light intensity 500-1000lx, and light intensity 9-13 h / d.

6. The method according to claim 1, characterized in that, The cultivation conditions for steps b and c are: cultivation temperature 22-27℃, light intensity 1000-2000lx, and light intensity 9-13 h / d.

7. The method according to claim 1, characterized in that, The matrix in step d is a mixed matrix of peat soil and perlite in a volume ratio of (2-4):(1-3).

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