A tissue culture rapid propagation method of caladium bicolor

Abstract

The application provides a tissue culture rapid propagation method of a colorful leaf taro plant, and relates to the technical field of plant tissue culture. The tissue culture rapid propagation method of the colorful leaf taro plant solves the technical pain points of high contamination rate, low proliferation coefficient, low survival rate of seedling raising and easy degradation of leaf color in the existing tissue culture of the colorful leaf taro by optimizing the pretreatment scheme of explants, customizing the medium formula in stages, dynamically controlling the culture environment and innovating the seedling raising and domestication process. The tissue culture method has the advantages of contamination rate ≤ 5%, proliferation coefficient 5-6 times, survival rate of seedling raising ≥ 90%, and more than 30% improvement of the stability of the color of the leaves of seedlings, and the growth cycle is shortened by 15-20 days. The method is suitable for multiple colorful leaf taro varieties such as apple flower, white lover and pink splashing, and has practical value for large-scale propagation.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for rapid tissue culture propagation of Caladium plants. Background Technology

[0002] Caladium bicolor, also known as variegated caladium, is a perennial herbaceous plant belonging to the genus Caladium in the family Araceae. Its brightly colored leaves and unique patterns make it a highly ornamental indoor foliage plant and landscaping plant. Traditional propagation methods mainly involve division and tuber propagation, but these methods suffer from low propagation coefficients, slow speed, and easy segregation of traits in offspring, making it difficult to meet the needs of large-scale production.

[0003] Plant tissue culture technology provides an effective way for the rapid propagation of caladium, but existing technologies still have many shortcomings: 1. Incomplete disinfection of explants often results in a contamination rate of over 30%, especially for tuber explants which are prone to carrying endophytic bacteria; 2. The culture medium formula is simple and mostly uses conventional hormone combinations, resulting in low adventitious bud induction rate, proliferation coefficient of only 2-3 times, and weak seedling growth; 3. The color of Caladium leaves depends on environmental regulation, and problems such as color fading and blurred patterns are prone to occur during tissue culture. 4. During the hardening-off and acclimatization stage, the seedlings have poor adaptability, fragile root systems, and a survival rate generally below 60%.

[0004] Therefore, developing a tissue culture method for caladium that is low in pollution, high in proliferation, high in survival rate, and can maintain the stability of leaf color has important application value. Summary of the Invention

[0005] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a rapid tissue culture propagation method for caladium plants. By optimizing explant pretreatment, customizing culture media in stages, dynamically controlling the culture environment, and innovating the hardening-off process, it achieves large-scale propagation with low pollution, high proliferation, and high survival rate, while maintaining the unique leaf color characteristics of caladium, thus solving the problems mentioned in the background technology.

[0006] (II) Technical Solution To achieve the above objectives, the present invention provides a method for rapid tissue culture propagation of Caladium plants, comprising the following steps: Step 1: Explant pretreatment: Select the lateral buds of the tubers with buds from the Caladium, remove the outer scales, perform pretreatment, and finally rinse with sterile water 5-6 times. Cut 2-3cm pieces of tissue with buds. Step 2: Induction Culture Pretreated explants were inoculated into an induction medium to induce adventitious shoot differentiation. Step 3: Proliferation Culture The induced adventitious buds were divided into 2-3 buds / clumps and inoculated into proliferation medium for proliferation culture. Step 4: Rooting Culture Select healthy seedlings that are 2-3 cm tall and inoculate them into rooting medium for rooting culture. Step 5: Seedling hardening and acclimatization: Place the rooted seedlings in a greenhouse (temperature 20-28℃, humidity 70-80%) for 3-5 days to harden them off. Open the bottle cap and continue hardening off for another 2-3 days. Transplant them into a substrate made of humus, perlite and coconut coir in a 2:1:1 ratio. Water thoroughly with a solution of 0.1% carbendazim to help them establish roots. Cover with a shade net (60% shading rate) and maintain a humidity of 75-85%. Cultivate for 15-20 days.

[0007] Preferably, in step one, the pretreatment is as follows: soaking in 75% alcohol for 30-45 seconds, disinfecting with 0.1% sodium hypochlorite solution (with 0.05% Tween-80 added) by shaking for 12-15 minutes, and disinfecting with 0.05% mercuric chloride solution for 3-5 minutes.

[0008] Preferably, in step two, the conditions of the induction medium are: light intensity of 1500-2000 lux, light duration of 12 h / d, and temperature of 23-25℃ for 25-30 days; the induction medium is based on MS medium, with the addition of 6-BA 1.5-2.0 mg / L, NAA 0.2-0.3 mg / L, humic acid 0.5-0.8 g / L, vitamin C 100-150 mg / L, sucrose 30 g / L, and agar 7 g / L, maintaining a pH of 5.8-6.0.

[0009] Preferably, in step three, the conditions of the proliferation medium are: light intensity of 2000-2500 lux, light duration of 14 h / d, and temperature of 22-24℃ for 20-25 days; the proliferation medium is based on MS medium, with the addition of 6-BA 1.0-1.2 mg / L, KT 0.3-0.5 mg / L, NAA 0.1-0.2 mg / L, banana puree 50 g / L, activated carbon 1 g / L, sucrose 30 g / L, and agar 7 g / L, maintaining a pH of 5.8-6.0.

[0010] Preferably, in step four, the conditions of the rooting medium are: light intensity of 2500-3000 lux, light duration of 16 h / d, and temperature of 23-25℃ for 15-20 days; the rooting medium is based on 1 / 2 MS medium, with the addition of IBA 0.8-1.0 mg / L, NAA 0.1 mg / L, humus extract 100 mL / L, sucrose 20 g / L, and agar 6.5 g / L, maintaining a pH of 5.8-6.0.

[0011] Preferably, in step one, the explants are selected in spring, from March to April, and the lateral buds of the tubers must be free from pests and diseases, have plump buds, and have a diameter of 0.5-0.8 cm.

[0012] Preferably, the humic acid is mineral-derived humic acid, and vitamin C is added after the culture medium is sterilized and cooled to 50-60°C.

[0013] Preferably, the banana puree is a homogenized paste made by peeling ripe bananas, and the activated carbon has a particle size of 100-200 mesh.

[0014] Preferably, the preparation method of the humus extract is as follows: humus and sterile water are mixed at a mass-volume ratio of 1:5, boiled for 30 minutes, filtered, and the supernatant is sterilized for later use.

[0015] Preferably, in step five, the substrate is sterilized by high-pressure sterilization at 121℃ for 30 minutes or by sun exposure for 3-5 days before transplanting.

[0016] (III) Beneficial Effects This invention provides a method for rapid tissue culture propagation of caladium plants, which has the following beneficial effects: 1. Through a three-level disinfection program, the explant contamination rate is ≤5%, which is far lower than the 30% or more of existing technologies; and the proliferation coefficient is 5-6 times, which is more than 80% higher than the traditional method (2-3 times), and the seedlings are robust and grow uniformly.

[0017] 2. The survival rate of seedlings after hardening is ≥90%, which solves the problem of low survival rate in traditional hardening methods. Furthermore, through light regulation and nutrient optimization, the seedling leaves are brightly colored and have clear patterns, and the color degradation rate is reduced by more than 30%. In addition, it only takes 90-100 days from explant induction to completion of hardening and transplanting, which is 15-20 days shorter than the traditional method, making it suitable for large-scale production. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the tissue culture rapid propagation method of the present invention; Figure 2 This is a schematic diagram of apple blossom tissue culture seedlings according to Embodiment 1 of the present invention; Figure 3 This is a schematic diagram of an apple blossom leaf according to Embodiment 1 of the present invention; Figure 4 This is a schematic diagram of an apple blossom leaf according to Embodiment 1 of the present invention; Figure 5 This is a schematic diagram of tissue culture seedlings of *White Lover*, a second embodiment of the present invention. Figure 6 This is a schematic diagram of the leaf of the White Lover plant, a second embodiment of the present invention. Figure 7 This is a schematic diagram of the leaf of the White Lover plant, a second embodiment of the present invention. Figure 8 This is a schematic diagram of pink splash tissue culture seedlings in Embodiment 3 of the present invention; Figure 9 This is a schematic diagram of the pink splash blade in Embodiment 3 of the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] The core technical solution of this invention is as follows: 1. Optimization of explant pretreatment: Select lateral buds of tubers (plump and free from pests and diseases) in March-April, and use a three-level disinfection scheme of "alcohol + sodium hypochlorite + mercuric chloride" to enhance the disinfection effect. At the same time, control the disinfection time to avoid damage to the explants and effectively reduce contamination by endophytic bacteria and surface bacteria.

[0021] 2. Staged culture medium formulation: (1) Induction medium: Adding humic acid enhances the stress resistance of explants, vitamin C inhibits browning, and 6-BA and NAA synergistically promote adventitious shoot differentiation, thus solving the problem of low induction rate in traditional methods; (2) Growth medium: Add KT to increase the growth coefficient, banana puree provides natural nutrients, activated carbon adsorbs metabolic waste, avoids excessive growth of seedlings, and makes the growth coefficient reach 5-6 times; (3) Rooting medium: 1 / 2MS was used to reduce the salt concentration and humus extract was added to simulate the natural growth environment, promote robust root system and improve transplant survival rate.

[0022] 3. Dynamic cultivation environment control: Adjust the light intensity (1500-3000 lux) and light duration (12-16 h / d) according to different cultivation stages to meet the different needs of bud differentiation, proliferation and rooting, while keeping the temperature stable at 22-25℃ to avoid abnormal growth caused by temperature fluctuations. High-intensity light also helps leaf pigment synthesis and maintains color stability.

[0023] 4. Two-stage hardening and acclimatization: First, harden the seedlings in bottles to adapt to temperature and humidity, then open the bottles to harden the seedlings to adapt to the air environment. The transplanting substrate uses a ratio of humus soil: perlite: coconut coir = 2:1:1, which takes into account both air permeability and water retention. Combined with carbendazim root-setting water and shade net protection, the survival rate of hardened seedlings is greatly improved.

[0024] Specifically, the following three examples illustrate this point: Example 1: like Figure 1 As shown, this embodiment of the invention provides a method for rapid tissue culture propagation of caladium plants, specifically a method for rapid tissue culture propagation of apple blossoms, comprising the following steps: Step 1: Explant pretreatment: In mid-March 2024, healthy apple blossom tubers were selected, and lateral buds with buds (0.6 cm in diameter) were cut off. The outer scales were removed, and the tubers were soaked in 75% alcohol for 40 seconds, sterilized by shaking in 0.1% sodium hypochlorite solution (with 0.05% Tween-80 added) for 13 minutes, sterilized in 0.05% mercuric chloride solution for 4 minutes, rinsed 5 times with sterile water, and 2.5 cm bud tissue blocks were cut off. Step 2: Induction Culture: Inoculate into induction medium (MS + 6-BA 1.8 mg / L + NAA 0.25 mg / L + humic acid 0.6 g / L + vitamin C 120 mg / L + sucrose 30 g / L + agar 7 g / L, pH=5.9), light intensity 1800 lux, light duration 12 h / d, temperature 24℃, culture for 28 days, adventitious shoot induction rate reached 92%, no browning phenomenon; Step 3: Proliferation Culture: Divide adventitious buds into 2 buds / clump, and inoculate them into proliferation medium (MS + 6-BA 1.1mg / L + KT 0.4mg / L + NAA 0.15mg / L + banana puree 50g / L + activated carbon 1g / L + sucrose 30g / L + agar 7g / L, pH=5.9), light intensity 2200 lux, light duration 14h / d, temperature 23℃, culture for 22 days, the proliferation coefficient reached 5.5 times, and the bud height was 2.8cm; Step 4: Rooting Culture: Select robust seedlings and inoculate them into rooting medium (1 / 2 MS + IBA 0.9 mg / L + NAA 0.1 mg / L + humus extract 100 mL / L + sucrose 20 g / L + agar 6.5 g / L, pH=5.9), light intensity 2800 lux, light duration 16 h / d, temperature 24℃, culture for 18 days, the rooting rate reaches 100%, with an average of 6-8 roots per seedling, and root length 1.5-2 cm; Step 5: Hardening off and Acclimatization: Place the seedlings in a greenhouse (25℃, 75% humidity) for 4 days, then open the lid and continue hardening for another 3 days. Transplant them into a sterilized substrate of humus:perlite:coconut coir = 2:1:1, water thoroughly with 0.1% carbendazim solution, cover with a 60% shade net, maintain 80% humidity, and after 18 days of cultivation, the survival rate reaches 93%. The red veins and pink markings on the leaves are clear, with no color fading. Figure 2 , Figure 3 and Figure 4 As shown.

[0025] Example 2: like Figure 1 As shown in the figure, this invention provides a method for rapid tissue culture propagation of Caladium plants, specifically a method for rapid tissue culture propagation of *Caladium 'White Lover'*, comprising the following steps: Step 1: Explant pretreatment: In early April 2024, lateral buds (0.5cm in diameter) of white lover tubers were selected, soaked in 75% alcohol for 35 seconds, sterilized with 0.1% sodium hypochlorite solution (with 0.05% Tween-80 added) by shaking for 12 minutes, sterilized with 0.05% mercuric chloride solution for 3 minutes, rinsed 6 times with sterile water, and 2cm tissue blocks with buds were cut. Step 2: Induction Culture: Inoculate into induction medium (MS + 6-BA 1.5mg / L + NAA 0.2mg / L + humic acid 0.5g / L + vitamin C 100mg / L + sucrose 30g / L + agar 7g / L, pH=5.8), light intensity 1500 lux, light duration 12h / d, temperature 23℃, culture for 25 days, adventitious shoot induction rate reaches 90%; Step 3: Proliferation Culture: Inoculate into proliferation medium (MS + 6-BA 1.0 mg / L + KT 0.3 mg / L + NAA 0.1 mg / L + banana puree 50 g / L + activated carbon 1 g / L + sucrose 30 g / L + agar 7 g / L, pH=5.8), light intensity 2000 lux, light intensity 14 h / d, temperature 22℃, culture for 20 days, the proliferation coefficient reaches 5.0 times; Step 4: Rooting Culture: Inoculate into rooting medium (1 / 2 MS + IBA 0.8 mg / L + NAA 0.1 mg / L + humus extract 100 mL / L + sucrose 20 g / L + agar 6.5 g / L, pH=5.8), light intensity 2500 lux, light duration 16 h / d, temperature 23℃, culture for 15 days, the rooting rate reaches 100%; Step 5: Hardening off and Acclimatization: Harden off the seedlings in the bottle for 3 days, then harden off for 2 days with the cap off. After transplanting, cultivate for 15 days. The survival rate reaches 91%. The leaves have a large white area, are evenly distributed, and show good growth. Figure 5 , Figure 6 and Figure 7 As shown.

[0026] Example 3: like Figure 1 As shown in the figure, this invention provides a method for rapid tissue culture propagation of Caladium plants, specifically: a rapid tissue culture propagation method for pink splashing plants, including the following steps: Step 1: Explant pretreatment: In late March 2024, pink splashing tuber lateral buds (0.7cm in diameter) were selected, soaked in 75% alcohol for 45 seconds, sterilized with 0.1% sodium hypochlorite solution (with 0.05% Tween-80 added) by shaking for 15 minutes, sterilized with 0.05% mercuric chloride solution for 5 minutes, rinsed 5 times with sterile water, and 3cm tissue blocks with buds were cut. Step 2: Induction Culture: Inoculate into induction medium (MS + 6-BA 2.0 mg / L + NAA 0.3 mg / L + humic acid 0.8 g / L + vitamin C 150 mg / L + sucrose 30 g / L + agar 7 g / L, pH=6.0), light intensity 2000 lux, light duration 12 h / d, temperature 25℃, culture for 30 days, adventitious shoot induction rate reaches 95%; Step 3: Proliferation Culture: Inoculate into proliferation medium (MS + 6-BA 1.2 mg / L + KT 0.5 mg / L + NAA 0.2 mg / L + banana puree 50 g / L + activated carbon 1 g / L + sucrose 30 g / L + agar 7 g / L, pH=6.0), light intensity 2500 lux, light intensity 14 h / d, temperature 24℃, culture for 25 days, the proliferation coefficient reaches 6.0 times; Step 4: Rooting culture: Inoculate into rooting medium (1 / 2 MS + IBA 1.0 mg / L + NAA 0.1 mg / L + humus extract 100 mL / L + sucrose 20 g / L + agar 6.5 g / L, pH=6.0), light intensity 3000 lux, light intensity 16 h / d, temperature 25℃, culture for 20 days, the rooting rate reaches 100%; Step 5: Hardening off and Acclimatization: Harden off the seedlings in the bottle for 5 days, then harden off for 3 days with the cap off. After transplanting, cultivate for 20 days. The survival rate reaches 95%, the leaves have bright pink variegation, and the growth is vigorous. Figure 8 and Figure 9 As shown.

[0027] Secondly, the MS medium and 1 / 2MS medium used in this invention are conventional culture media for plant tissue culture, which can be purchased commercially or prepared by oneself; the humic acid used is mineral-derived humic acid (purity ≥90%), the vitamin C is analytical grade, and the banana puree is prepared from ripe, unrotten bananas; the light in the culture environment is provided by LED plant growth lamps, and the humidity is controlled by a humidifier to ensure stable culture conditions; this invention is applicable to a variety of caladium varieties, and in addition to the above-mentioned embodiments, it also has good propagation effects on varieties such as purple caladium and variegated caladium.

[0028] In summary, the embodiments of this invention address the technical challenges of high contamination rates, low proliferation coefficients, low seedling survival rates, and easy leaf color degradation in existing caladium tissue culture by optimizing explant pretreatment schemes, customizing staged culture medium formulations, dynamically controlling the culture environment, and innovating seedling hardening and acclimatization processes. The tissue culture method of this invention achieves a contamination rate of ≤5%, a proliferation coefficient of 5-6 times, a seedling survival rate of ≥90%, and improves seedling leaf color stability by more than 30%, while shortening the growth cycle by 15-20 days. It is applicable to various caladium varieties such as Apple Blossom, White Lover, and Pink Splash, and has practical value for large-scale propagation.

[0029] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for rapid tissue culture propagation of Caladium plants, characterized in that: Includes the following steps: Step 1: Explant pretreatment: Select Caladium ( Caladium bicolor The lateral buds of the tubers with buds are pretreated after removing the outer scales. Finally, they are rinsed with sterile water 5-6 times and 2-3 cm pieces of tissue with buds are cut off. The pretreatment is as follows: soaking in 75% alcohol for 30-45 seconds, shaking and disinfecting with 0.1% sodium hypochlorite solution for 12-15 minutes, and disinfecting with 0.05% mercuric chloride solution for 3-5 minutes. Step 2: Induction Culture Pretreated explants were inoculated into an induction medium to induce adventitious shoot differentiation. The conditions of the induction medium were: light intensity of 1500-2000 lux, light duration of 12 h / d, and temperature of 23-25℃ for 25-30 days. The induction medium consisted of MS basal medium, 6-BA 1.5-2.0 mg / L, NAA 0.2-0.3 mg / L, humic acid 0.5-0.8 g / L, vitamin C 100-150 mg / L, sucrose 30 g / L, and agar 7 g / L, maintaining a pH of 5.8-6.

0. Step 3: Proliferation Culture The induced adventitious buds were divided into 2-3 buds / clump and inoculated into a proliferation medium for proliferation culture. The conditions of the proliferation medium were: light intensity 2000-2500 lux, light duration 14h / d, and temperature 22-24℃ for 20-25 days. The proliferation medium consisted of MS basal medium, 6-BA 1.0-1.2mg / L, KT 0.3-0.5mg / L, NAA 0.1-0.2mg / L, banana puree 50g / L, activated carbon 1g / L, sucrose 30g / L, and agar 7g / L, maintaining a pH of 5.8-6.

0. The banana puree was a homogenized paste made from peeled mature bananas, and the activated carbon had a particle size of 100-200 mesh. Step 4: Rooting Culture Healthy seedlings with a height of 2-3 cm were selected and inoculated into a rooting medium for rooting culture. The conditions of the rooting medium were as follows: light intensity of 2500-3000 lux, light duration of 16 h / d, and temperature of 23-25℃ for 15-20 days. The rooting medium consisted of 1 / 2 MS basal medium, IBA 0.8-1.0 mg / L, NAA 0.1 mg / L, humus extract 100 mL / L, sucrose 20 g / L, and agar 6.5 g / L, maintaining a pH of 5.8-6.

0. The humus extract was prepared by mixing humus and sterile water at a mass-to-volume ratio of 1:5, boiling for 30 min, filtering, and sterilizing the supernatant for later use. Step 5: Seedling hardening and acclimatization: Place the rooted seedlings in the greenhouse with the bottle for 3-5 days to harden off, then open the bottle cap and continue hardening off for another 2-3 days. Transplant them into a substrate made of humus, perlite and coconut coir in a ratio of 2:1:

1. Water thoroughly with a solution of 0.1% carbendazim to help them establish roots. Cover with a shade net and maintain a humidity of 75-85%. Cultivate for 15-20 days.

2. The method for rapid tissue culture propagation of Caladium plants according to claim 1, characterized in that: In step one, the explants are selected in spring, from March to April. The lateral buds of the tubers must be free from pests and diseases, have plump buds, and have a diameter of 0.5-0.8cm.

3. The method for rapid tissue culture propagation of Caladium plants according to claim 1, characterized in that: The humic acid is mineral-derived humic acid, and vitamin C is added after the culture medium is sterilized and cooled to 50-60°C.

4. The method for rapid tissue culture propagation of Caladium plants according to claim 1, characterized in that: In step five, the substrate is sterilized at 121℃ for 30 minutes before transplanting, or disinfected by sun exposure for 3-5 days.

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