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) proliferation and rooting culture; and 4) transplanting test-tube plantlets. According to the method, the bud of the arrowroot is selected as the explant, the disinfection step is optimized, the improved MS culture medium is selected to replace a common MS culture medium, and explant adventitious bud induction, proliferation and rooting culture are carried out, so that the disinfection survival rate of the arrowroot, the induction rate of the adventitious bud, the proliferation rate of multiple shoots, the rooting rate of seedlings and the transplanting survival rate are remarkably increased; 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

Technical Field

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

[0002] Blue Rose Maranta, belonging to the genus *Calathea* of the family Marantaceae, is a popular foliage plant worldwide, also known as the "oil painting plant." A perennial herb of the Marantaceae family, it has broad application prospects due to its diverse varieties and vibrant leaf colors. Traditional methods of propagating Maranta plants typically include division and cuttings, but these have low propagation coefficients, resulting in a very limited number of seedlings. Therefore, tissue culture rapid propagation technology has become an important method for propagating Blue Rose Maranta. This invention patent uses the base of young shoots of Blue Rose Maranta 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 blue rose arrowroot 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 blue rose arrowroot tissue culture, characterized by comprising the following steps:

[0006] a. Disinfection and induction culture of explants: Select disease-free blue rose arrowroot mother plants and take newly grown trumpet-shaped shoots as explants. After digging the shoots from the ground, do not wash them with water. First, cut off the upper leaves and remove the outer bracts. Then, disinfect the 2-3 cm base of the stem, remove excess tissue, and inoculate them into the culture medium until adventitious shoots of the explants grow. The composition of the induction culture medium is: modified MS medium + 3.0-5.0 mg / L 6-BA + 0.1-0.2 mg / L NAA + 20-30 g / L sucrose + 5.8-7 g / L agar, pH 5.7-7;

[0007] a. Proliferation and rooting culture: The leaves and stems of the obtained adventitious buds are removed, and the base of the buds is inoculated into a proliferation and rooting medium for culture to form clustered buds with roots; the proliferation and rooting medium is: modified MS medium, supplemented with 2g / L of Flower Treasure No. 1, 100ml / L coconut juice + 2.0-4.0mg / L 6-BA + 0.1-0.3mg / L KT + 0.1-0.3mg / L NAA + 20-30g / L sucrose + 6-7g / L agar, pH 5.8-6.0;

[0008] b. 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.

[0009] The modified MS medium described in step a is a standard MS medium with the concentrations of ammonium nitrate adjusted to 1900 mg / L, potassium nitrate adjusted to 1500 mg / L, and magnesium sulfate adjusted to 450 mg / L, while the other components remain unchanged.

[0010] The modified MS medium described in step b is a standard MS medium with the following components adjusted: ammonium nitrate concentration adjusted to 1900 mg / L, potassium nitrate concentration adjusted to 1500 mg / L, and magnesium sulfate concentration adjusted to 450 mg / L, while the other components remain unchanged.

[0011] Preferably, the explants mentioned in step a are obtained through the following pretreatment method: Select new shoots of mature blue rose arrowroot as materials. 42 days before the explants are selected and disinfected, irrigate once with a 50% carbendazim wettable powder solution diluted 500-800 times 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 again after 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 blue rose arrowroot as explants.

[0012] Preferably, the disinfection is performed by 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.

[0013] Preferably, the method of disinfecting continuously with 0.1% mercuric chloride solution for several times specifically means disinfecting continuously with 0.1% mercuric chloride solution for 3 times, with the first disinfection time being 4-6 minutes, the second disinfection time being 3-5 minutes, and the third disinfection time being 1-3 minutes.

[0014] Preferably, the cultivation conditions for step a are: cultivation temperature 22-27℃, light intensity 500-1000lx, and light intensity 9-13 h / d.

[0015] Preferably, the cultivation conditions in step b are: cultivation temperature 22-27℃, light intensity 1000-2000lx, and light intensity 9-13 h / d.

[0016] Preferably, the matrix in step c is a mixed matrix of peat moss and perlite in a volume ratio of (2-4):(1-3), and more preferably 3-4:1-2.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. Growth control of arrowroot regeneration: By adjusting the concentration of basal culture medium, plant growth hormones and growth promoting factors, precise control of arrowroot tissue growth can be achieved, which helps to optimize the regeneration process.

[0019] 2. Propagation and breeding: Using the culture medium of the present invention, blue rose calathea can be rapidly propagated to produce new plants with the same genetic characteristics. Plants regenerated through tissue culture can maintain the genetic characteristics of the parents, which is very beneficial for propagation and breeding projects and helps to cultivate new calathea varieties.

[0020] 3. In summary, this invention provides an effective tool for the in vitro regeneration of arrowroot, which helps to accelerate the propagation and breeding process of blue rose arrowroot, improve the controllability of growth conditions, reduce costs, and contribute to the realization of sustainable arrowroot cultivation. Attached Figure Description

[0021] Figure 1 This is a rapid propagation method for blue rose arrowroot tissue culture, where A: adventitious shoots induced on modified MS medium; B: differentiation of adventitious shoots into seedlings; and C: rooting during the induction process. Detailed Implementation

[0022] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0023] Example 1:

[0024] 1. Explant sterilization and adventitious bud induction: Blue Rose Maranta explants 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 pest-free superior Blue Rose Maranta 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 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 chlorothalonil-fludioxonil solution, select the treated Blue Rose Maranta new shoots as explants for tissue culture.

[0025] 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 75% (modified MS medium). 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) + 3.0 mg / L 6-BA + 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.

[0026] Experimental Results: Different basal culture media had significant effects on the induction of adventitious shoots in *Maranta purpurea*. With MS as the basal medium, the induction rate of explants was 85.97%, while with modified MS as the basal medium, the induction rate reached 92.50% (20 days post-inoculation), which was 6.53% higher than that with MS as the basal medium. Furthermore, shoot germination could be observed 5-7 days post-inoculation. Figure 1 A) Germination time is 2-3 days earlier than that of MS basal medium. Therefore, the modified MS medium was selected as the basal medium with the best explant induction effect.

[0027] 2. Proliferation and Rooting Culture: Adventitious buds induced by the above-mentioned modified MS medium as the basal medium were defoliated and their stems removed. The basal parts of the buds were inoculated onto the proliferation and rooting medium for culture. The culture temperature was 24℃, the light intensity was 1000lx, and the photoperiod was 13h / d. The proliferation and rooting induction medium was as follows: MS medium (control) or modified MS medium as the basal medium (the concentration of ammonium nitrate in the ordinary MS medium formula was adjusted to 1900mg / L, the concentration of potassium nitrate was adjusted to 1500mg / L, and the concentration of magnesium sulfate was adjusted to 450mg / L, while the other components remained unchanged), with the addition of 2g of Flower Treasure No. 1, 100ml coconut juice + 2.0 mg / L 6-BA + 0.2 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 value, and sterilize for later use.

[0028] Experimental Results: Using 45 days as one subculture cycle, after four consecutive cultures, different basal media showed significant differences in their effects on the proliferation and rooting of *Calathea speciosa*. With MS as the basal medium, the proliferation rate of *Calathea speciosa* was 2.3 times, with no rooting observed; while with the modified MS medium as the basal medium, clustered shoots formed, with each seedling bearing 2-3 roots, and proliferation and rooting were completed in one step, achieving a proliferation rate of 4.5 times. Figure 1 The proliferation rate of B and C media was 2.2 times higher than that of MS basal medium. Therefore, the modified MS medium was selected as the most suitable basal medium for proliferation and rooting culture.

[0029] 3. Transplanting of test-tube seedlings: When the modified MS medium used as the basal medium has grown to 5 cm in height, transfer the seedlings to natural light for 3 days to harden them off. Then, open the stopper and use tweezers to remove the 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 ).

[0030] Example 2:

[0031] 1. Explant sterilization and adventitious bud induction: The explants of *Calathea rosea* carry dust, microorganisms, and endophytic bacteria, requiring thorough sterilization without damaging plant cells. Therefore, successful explant sterilization is the first step in tissue culture. During the budding season, select vigorous, disease-free *Calathea rosea* strains grown in greenhouses. 42 days before inoculation, drench the plants once with a 600-fold dilution of 50% carbendazim wettable powder and spray the leaves. Seven days later, drench the plants once with a 4000-fold dilution of agricultural fungicide-fludioxonil soluble liquid and spray the leaves. Repeat this process for another seven days, for a total of four treatments. Seven days after the final drench with the fungicide-fludioxonil solution, select the treated *Calathea rosea* shoots as explants for tissue culture.

[0032] Newly sprouted trumpet-shaped shoots of *Calathea purpurea* 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 (explant induction rate) of 93% (data at 20 days), and a disinfection success rate of 88%. The induction medium consists of: 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 + 0.15 mg / L NAA + 25 g / L sucrose + 6.5 g / L agar, pH 5.8. The preparation method is to add each component to the basic medium, adjust the pH value, and sterilize for later use.

[0033] 2. Proliferation and Rooting Culture: Leaves and stems were removed from the induced adventitious buds, and the basal parts of the buds were inoculated onto a proliferation and rooting medium for culture, forming clustered buds. Each seedling was a complete plant with 2-3 roots. The culture temperature was 24℃, the light intensity was 1500 lx, and the photoperiod was 11 h / d. One subculture proliferation cycle was 45 days. After four consecutive culture cycles, the proliferation ratio after each subculture proliferation cycle was 4.8 times. The proliferation and rooting induction medium is as follows: the basal medium is 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), with the addition of 2 g / L of Flower Treasure No. 1 coconut juice 100 ml + 3.0 mg / L 6-BA + 0.2 mg / L KT + 0.2 mg / L NAA + 25 g / L sucrose + 6.5 g / L agar, pH 5.9. The preparation method is to add each component to the basal medium, adjust the pH value, and sterilize for later use.

[0034] 3. Transplanting of test-tube seedlings: When the proliferated rooted test-tube seedlings grow to 5cm in height, transfer them to natural light for 5 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 4:2 by volume. Pay attention to watering, shading, keeping warm and moist. The survival rate can reach 98%.

[0035] Example 3:

[0036] 1. Explant sterilization and adventitious bud induction: The explants of *Calathea rosea* carry dust, microorganisms, and endophytic bacteria, requiring thorough sterilization without damaging plant cells. Therefore, successful explant sterilization is the first step in tissue culture. During the budding season, select vigorous, disease-free *Calathea rosea* strains grown in greenhouses. 42 days before inoculation, drench the plants once with a 600-fold dilution of 50% carbendazim wettable powder and spray the leaves. Seven days later, drench the plants once with a 4000-fold dilution of agricultural fungicide-fludioxonil soluble liquid and spray the leaves. Repeat this process for another seven days, for a total of four treatments. Seven days after the final drench with the fungicide-fludioxonil solution, select the treated *Calathea rosea* shoots as explants for tissue culture.

[0037] Newly sprouted trumpet-shaped shoots of *Calathea purpurea* 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 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, and disinfected with a 0.1% mercuric chloride solution for 4 minutes, rinsed 4 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 1000 lx, and the light duration was 13 h / d. Adventitious buds formed after 15 days of culture, with an adventitious bud occurrence rate (explant induction rate) of 88% (data at 20 days). The disinfection success rate was 86%, and some plants showed browning. The induction medium consists of: 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) + 5.0 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 7.0 g / L agar, pH 5.9. The preparation method is to add each component to the basal medium, adjust the pH value, and sterilize for later use.

[0038] 2. Proliferation and Rooting Culture: Leaves and stems were removed from the induced adventitious buds, and the basal parts of the buds were inoculated onto a proliferation and rooting medium for culture, forming clustered buds. Each seedling was a complete plant with 2-3 roots. The culture temperature was 24℃, the light intensity was 2000 lx, and the photoperiod was 9 h / d. One subculture proliferation cycle was 45 days. After four consecutive culture cycles, the proliferation ratio after each subculture proliferation cycle was 4.4 times. The proliferation and rooting induction medium is as follows: the basal medium is 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), with the addition of 2g of Flower Treasure No. 1, 100ml of coconut juice + 4.0 mg / L 6-BA + 0.2 mg / L KT + 0.3 mg / L NAA + 30 g / L sucrose + 7.0 g / L agar, pH 5.9. The preparation method is to add each component to the basal medium, adjust the pH value, and sterilize for later use.

[0039] 3. Transplanting of test-tube seedlings: When the proliferated rooted test-tube seedlings grow to 5cm in height, transfer 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 94%.

[0040] 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 Blue Rose Maranta through tissue culture, characterized in that, Includes the following steps: a. Disinfection and induction culture of explants: Select disease-free blue rose arrowroot mother 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 2-3 cm stem base, remove excess tissue, and inoculate them in the induction medium until adventitious shoots grow from the explants. The composition of the induction medium is: modified MS medium + 3.0-5.0 mg / L 6-BA + 0.1-0.2 mg / L NAA + 20-30 g / L sucrose + 5.8-7 g / L agar, pH 5.7-7; b. Proliferation and rooting culture: The leaves and stems of the obtained adventitious buds are removed, and the base of the buds is inoculated into a proliferation and rooting medium for culture to form clustered buds with roots; the proliferation and rooting medium is: modified MS medium, supplemented with 2g / L of Flower Treasure No. 1, 100ml / L coconut juice + 2.0-4.0mg / L 6-BA + 0.1-0.3mg / L KT + 0.1-0.3mg / L NAA + 20-30g / L sucrose + 6-7g / L agar, pH 5.8-6.0; c. 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. The modified MS medium described in step a is a standard MS medium with the concentrations of ammonium nitrate adjusted to 1900 mg / L, potassium nitrate adjusted to 1500 mg / L, and magnesium sulfate adjusted to 450 mg / L, while the other components remain unchanged. The modified MS medium described in step b is a standard MS medium with the following components adjusted: ammonium nitrate concentration adjusted to 1900 mg / L, potassium nitrate concentration adjusted to 1500 mg / L, and magnesium sulfate concentration adjusted to 450 mg / L, while the other components remain 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 blue rose arrowroot as materials. 42 days before the explants were selected and disinfected, irrigate once with a 50% carbendazim wettable powder solution diluted 500-800 times and spray the leaves. 7 days later, irrigate once with a 3500-4500 times diluted solution of agricultural cyprodinil·fludioxonil soluble liquid and spray the leaves. Repeat the above steps again after another 7 days, for a total of 4 treatments. 7 days after the last irrigation with cyprodinil·fludioxonil aqueous solution, select the treated blue rose arrowroot new shoots as explants.

3. The method according to claim 2, characterized in that, The disinfection process involves wiping the surface of the explant with cotton balls 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 to obtain the disinfected explant.

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 4-6 minutes, the second disinfection time is 3-5 minutes, and the third disinfection time is 1-3 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 step b 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 c is a mixed matrix of peat soil and perlite in a volume ratio of (2-4): (1-3).

8. The method according to claim 7, characterized in that, The matrix in step c is peat moss and perlite in a volume ratio of 3-4:1-2.

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