Tissue culture and rapid propagation method for colocasia esculenta plants

Abstract

The invention provides a tissue culture and rapid propagation method for colocasia esculenta plants, and belongs to the technical field of plant tissue culture. The method comprises the following steps: by taking stems or leaves of colocasia esculenta plants as explants, disinfecting, inoculating into a callus induction culture medium, and culturing to obtain calluses; and dispersing the calluses by adopting a mashed garlic mashing method, inoculating the calluses into a proliferation culture medium, and carrying out proliferation culture. By optimizing the components and composition of the callus induction culture medium and adopting a mashed garlic mashing method for proliferation culture, the proliferation coefficient of the colocasia esculenta is increased to 30 times, a large number of tissue culture seedlings can be obtained in a short time by using a small space and a small number of female parents, time and labor are saved, and technical support is provided for propagation and popularization of the colocasia esculenta seedlings.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to a tissue culture rapid propagation method for caladium plants. Background Art

[0002] Caladium Vent. is a perennial herbaceous plant of the Araceae family. It is also known as variegated taro and colorful taro. It is native to tropical South America and has a variety of colors, including red, white, pink, yellow, purple, and red and green. It is one of the most colorful foliage plants with rich leaf color changes and is known as the "Queen of Leaf Viewing". Summer is its main viewing period, and the colorful leaves are full of coolness; the leaves gradually become messy in autumn, and the leaves turn yellow in winter and enter a dormant period, and begin to sprout and grow again in late spring and early summer; May to September is its vigorous growth period; the leaves of Caladium Vent. are shaped like elephant ears, colorful, and eye-catching, making it a top-grade foliage plant.

[0003] The conventional propagation method of caladium is division or bulb division, which can be carried out throughout the year, but the proliferation rate is very low. Commercial propagation can also be carried out through seeds, but seed propagation is difficult because the seeds are very small and have a high mortality rate. The cost of plants obtained by seed growth and propagation is too high. In addition, the offspring of seed propagation are very prone to mutation and degeneration, and it is difficult to maintain the original traits.

[0004] The existing tissue culture method of colored leaf caladium adopts the method of dividing callus into groups for cultivation, which has problems such as inconsistent growth rate, uneven growth of culture, and low reproduction efficiency. Therefore, how to establish a more efficient and rapid tissue culture and reproduction method for colored leaf caladium plants to facilitate commercialization and industrial production is an urgent problem that technicians in this field need to solve. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a tissue culture rapid propagation method for caladium plants, so as to solve the problems of inconsistent growth rate, uneven culture growth, low propagation efficiency, etc. in the cultivation process of existing propagation methods.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a tissue culture rapid propagation method for caladium plants, comprising the following steps:

[0008] 1) using stems or leaves of caladium plants as explants, sterilizing them, and inoculating them into callus induction medium for culture to obtain callus;

[0009] 2) The callus tissue is dispersed by mashing garlic and then inoculated into a proliferation culture medium for proliferation culture.

[0010] Preferably, the callus induction medium in step 1) is based on MS medium and further includes components with the following concentrations: 2 - 4 mg / L 6-BA, 0.5 - 1.5 mg / L TDZ, 0.05 - 0.15 mg / L IBA, 25 - 35 g / L sucrose, and 3 - 7 g / L carrageenan.

[0011] Preferably, the temperature of the culture in step 1) is 23 - 27 °C. The culture methods include dark culture and light culture. The time of dark culture is 5 - 9 d, the light intensity of light culture is 40 - 50 μmol·m -2 ·s -1 , the light time of light culture is 10 - 14 h / d, and the time of light culture is 21 - 25 d.

[0012] Preferably, the proliferation medium in step 2) is based on MS medium and further includes components with the following concentrations: 2 - 4 mg / L 6-BA, 0.5 - 1.5 mg / L TDZ, 0.05 - 0.15 mg / L IBA, 25 - 35 g / L sucrose, and 3 - 7 g / L carrageenan.

[0013] Preferably, the proliferation culture method in step 2) is light culture. The light intensity of proliferation culture is 40 - 50 μmol·m -2 ·s -1 , the light time of proliferation culture is 10 - 14 h / d, the temperature of proliferation culture is 23 - 27 °C, and the time of proliferation culture is 18 - 22 d.

[0014] Preferably, the length of the explant is 0.8 - 1.2 cm.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] By optimizing the components and composition of the callus induction medium and adopting the method of mashing with garlic puree for proliferation culture, the proliferation coefficient of Caladium bicolor is increased to 30 times. It can utilize a very small space and obtain a large number of tissue culture seedlings from very few mother plants in a short time, saving time and labor, and providing technical support for the propagation and popularization of Caladium bicolor seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the mother plant selected for the explant;

[0018] Figure 2 is the disinfected leaf;

[0019] Figure 3 are the calluses formed after the leaves are induced by different callus induction media;

[0020] Figure 4 It is the callus cutting and proliferation culture process by the ordinary method;

[0021] Figure 5 It is the callus cutting and proliferation culture process by the garlic puree pounding method. Specific implementation mode

[0022] The present invention provides a rapid tissue culture and propagation method for caladium plants, comprising the following steps:

[0023] 1) Using the stem or leaf of caladium plants as explants, after disinfection, inoculating them into the callus induction medium for culture to obtain callus;

[0024] 2) Dispersing the callus by the garlic puree pounding method and then inoculating it into the proliferation medium for proliferation culture.

[0025] In the present invention, using the stem or leaf of caladium plants as explants, after disinfection, inoculating them into the callus induction medium for culture to obtain callus. The length of the explants is preferably 0.8 - 1.2 cm, more preferably 0.9 - 1.1 cm, and even more preferably 1 cm; washing in dishwashing detergent water before disinfection; sequentially using alcohol solution and mercuric chloride solution for disinfection, the volume fraction of the alcohol solution is preferably 70 - 80%, more preferably 72 - 78%; the alcohol disinfection time is preferably 8 - 12 s, more preferably 9 - 11 s; the volume fraction of the mercuric chloride solution is preferably 0.05 - 0.15%, more preferably 0.08 - 0.12%; the mercuric chloride solution disinfection time is preferably 6 - 10 min, more preferably 7 - 9 min. The callus induction medium is based on MS medium and also includes the following components in concentrations: 2 - 4 mg / L 6 - BA, 0.5 - 1.5 mg / L TDZ, 0.05 - 0.15 mg / L IBA, 25 - 35 g / L sucrose, and 3 - 7 g / L carrageenan; the concentration of 6 - BA is preferably 2.5 - 3.5 mg / L; the concentration of TDZ is preferably 0.8 - 1.2 mg / L; the concentration of IBA is preferably 0.08 - 0.12 mg / L; the concentration of sucrose is preferably 28 - 32 g / L; the concentration of carrageenan is preferably 4 - 6 g / L; the pH of the callus induction medium is preferably 5.5 - 6.2, more preferably 5.7 - 6.0; the culture temperature is preferably 23 - 27 °C, more preferably 24 - 26 °C; the culture method is dark culture and light culture, the dark culture time is preferably 5 - 9 d, more preferably 6 - 8 d; the light intensity of the light culture is preferably 40 - 50 μmol·m -2 ·s -1 , more preferably 42 - 48 μmol·m-2 ·s -1 ; The light exposure time for the light cultivation is preferably 10 - 14 h / d, more preferably 11 - 13 h / d; The time for the light cultivation is preferably 21 - 25 d, more preferably 22 - 24 d.

[0026] In the present invention, the callus is dispersed by the garlic puree mashing method and then inoculated into the proliferation medium for proliferation culture. The proliferation medium is based on MS medium and further includes the following components at the following concentrations: 2 - 4 mg / L 6 - BA, 0.5 - 1.5 mg / L TDZ, 0.05 - 0.15 mg / L IBA, 25 - 35 g / L sucrose, and 3 - 7 g / L carrageenan; The concentration of 6 - BA is preferably 2.5 - 3.5 mg / L; The concentration of TDZ is preferably 0.8 - 1.2 mg / L; The concentration of IBA is preferably 0.08 - 0.12 mg / L; The concentration of sucrose is preferably 28 - 32 g / L; The concentration of carrageenan is preferably 4 - 6 g / L; The pH of the proliferation medium is preferably 5.5 - 6.2, more preferably 5.7 - 6.0. The mode of the proliferation culture is preferably light cultivation, and the light intensity of the proliferation culture is preferably 40 - 50 μmol·m -2 ·s -1 , more preferably 42 - 48 μmol·m -2 ·s -1 ; The light exposure time for the proliferation culture is preferably 10 - 14 h / d, more preferably 11 - 13 h / d; The temperature of the proliferation culture is preferably 23 - 27 °C, more preferably 24 - 26 °C; The time for the proliferation culture is preferably 18 - 22 d, more preferably 19 - 21 d.

[0027] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0028] Example 1

[0029] Select potted caladium with good growth, first use Nongdi Le diluted with 1000 times water for root irrigation to eliminate the eggs in the matrix, and then use Guoguang Yingna diluted with 2000 times water for leaf spray sterilization. Dry water treatment, after the water is completely dry, select annual leaves without diseases and insects as explants, take the leaves of the newly cut caladium, put them in detergent water and brush them lightly with a brush, and rinse them in running tap water for 0.5h. Rinse the leaves with sterile water 3 times, then disinfect them with 75% alcohol for 10s, rinse them with sterile water 3 times, disinfect them with 0.1% mercuric chloride for 8min, and finally rinse them with sterile water 6 times (shake them by hand for 1min each time with sterile water), and use sterile filter paper to absorb the surface moisture. Cut the disinfected leaves into 1cm small sections with a surgical blade on the clean bench, and cut off part of the cut surface tissue.

[0030] The explants were inoculated into callus induction medium (MS-based medium, composed of the following components: 3 mg / L 6-BA, 1 mg / L TDZ, 1 mg / L IBA, 30 g / L sucrose and 5 g / L carrageenan, pH 5.8) and placed in a culture room for culture at a light intensity of 45 μmol·m -2 ·s -1 The first week was dark culture (no light culture), and one week later, light culture was performed with a light duration of 12 h / d and a culture temperature of 25°C. Callus tissue was obtained after 30 days of culture.

[0031] The callus tissue was cut into small balls with a diameter of 0.2 cm and placed in a proliferation medium (MS-based medium consisting of the following components: 3 mg / L 6-BA, 1 mg / L TDZ, 0.1 mg / L IBA, 30 g / L sucrose and 5 g / L carrageenan, pH 5.8) and cultured in a culture room under light conditions with an illumination of 45 μmol·m -2 ·s -1 The culture medium was set at 25°C and the illumination time was 12 h / d. The callus was cultured for 20 days. When the small clusters grew into 0.5 cm clumps, the small clusters were taken out with tweezers and dispersed in a pestle using garlic mashing method. The dispersed callus was cultured in a proliferation medium (MS as the basic medium, composed of the following concentrations: 3 mg / L 6-BA, 1 mg / L TDZ, 0.1 mg / L IBA, 30 g / L sucrose and 5 g / L carrageenan, pH 5.8).

[0032] Example 2

[0033] Select well-growing Caladium bicolor potted plants. First, irrigate the roots with Nongdile diluted 1000 times to eliminate the insect eggs in the substrate, and then spray the leaves with Guoguang Yinna diluted 2000 times for bactericidal treatment. For dry treatment, after the water is completely dry, select one-year-old leaves without pests and diseases as explants. Take the leaves of the newly cut Caladium bicolor, put them into the dishwashing liquid water, gently brush them clean with a brush, and rinse them under running tap water for 0.5 h. After rinsing the leaves 3 times with sterile water, disinfect them with 70% alcohol for 8 s, then rinse them 2 times with sterile water, disinfect them with 0.05% mercuric chloride for 6 min, and finally rinse them 5 times with sterile water (shake them manually for 0.5 min each time during the above sterile water rinsing). Blot the surface moisture with sterile filter paper. On the ultra-clean workbench, cut the disinfected leaves into 0.8 cm segments with a surgical blade, and cut off the tissue at the cut surface.

[0034] Inoculate the explants into the callus induction medium (using MS as the basic medium, composed of the following components at the following concentrations: 2 mg / L 6-BA, 0.5 mg / L TDZ, 0.05 mg / L IBA, 25 g / L sucrose, and 3 g / L carrageenan, pH 5.5). After inoculation, place them in the culture room for cultivation. The light intensity is 40 μmol·m -2 ·s -1 , and conduct dark cultivation (without light cultivation) for the first 5 days. After 5 days, conduct light cultivation. The light time is 10 h / d, the cultivation temperature is 23 °C, and callus is obtained after 26 days of cultivation.

[0035] Cut the callus into small masses with a diameter of 0.1 cm, and place them in the proliferation medium (using MS as the basic medium, composed of the following components at the following concentrations: 2 mg / L 6-BA, 0.5 mg / L TDZ, 0.05 mg / L IBA, 25 g / L sucrose, and 3 g / L carrageenan, pH 5.5). Cultivate them under the light conditions in the culture room. The light intensity is 40 μmol·m -2 ·s -1 , the light is 10 h / d, the temperature is 23 °C, and cultivate for 18 days. Wait until the small masses grow into masses with a size of 0.4 cm. Take out the small masses with forceps and disperse them in a mortar using the garlic puree method. Proliferate and culture the dispersed callus in the proliferation medium (using MS as the basic medium, composed of the following components at the following concentrations: 2 mg / L 6-BA, 0.5 mg / L TDZ, 0.05 mg / L IBA, 25 g / L sucrose, and 3 g / L carrageenan, pH 5.5).

[0036] Example 3

[0037] Select well-growing caladium potted plants. First, irrigate the roots with Nongdile diluted 1000 times to eliminate the eggs in the substrate, and then spray the leaves with Guoguang Yinna diluted 2000 times for disinfection. After air-drying, select one-year-old leaves without pests and diseases as explants when the water is completely dry. Take the leaves of the newly cut caladium, gently brush them clean with a writing brush in dishwashing liquid water, and rinse them under running tap water for 0.5 h. Rinse the leaves 3 times with sterile water, disinfect them with 80% alcohol for 12 s, then rinse them 3 times with sterile water, disinfect them with 0.15% mercuric chloride for 10 min, and finally rinse them 7 times (shake them manually for 1.5 min each time during the above-mentioned sterile water rinsing), and blot the surface moisture with sterile filter paper. On the ultra-clean workbench, cut the disinfected leaves into 1.2 cm small segments and cut off the tissue at the cut surface part.

[0038] Inoculate the explants on the callus induction medium (with MS as the basic medium, composed of the following components at the following concentrations: 4 mg / L 6-BA, 1.5 mg / L TDZ, 0.15 mg / L IBA, 35 g / L sucrose, and 7 g / L carrageenan, pH 6.2). After inoculation, place them in the culture room for cultivation. The light intensity is 50 μmol·m -2 ·s -1 , dark-culture for the first 9 days (cultivate without light), and then light-culture after 9 days. The light time is 14 h / d, the culture temperature is 27 °C, and callus is obtained after 34 days of cultivation.

[0039] Cut the callus into small masses with a diameter of 0.3 cm and place them in the proliferation medium (with MS as the basic medium, composed of the following components at the following concentrations: 4 mg / L 6-BA, 1.5 mg / L TDZ, 0.15 mg / L IBA, 35 g / L sucrose, and 7 g / L carrageenan, pH 6.2). Cultivate them under the light conditions in the culture room. The light intensity is 50 μmol·m -2 ·s -1 , the light is 14 h / d, the temperature is 27 °C, and cultivate for 22 days. Wait until the small masses grow into masses with a size of 0.6 cm, take out the small masses with forceps and disperse them in a masher by using the garlic puree mashing method. Proliferate and culture the dispersed callus in the proliferation medium (with MS as the basic medium, composed of the following components at the following concentrations: 4 mg / L 6-BA, 1.5 mg / L TDZ, 0.15 mg / L IBA, 35 g / L sucrose, and 7 g / L carrageenan, pH 6.2).

[0040] Experimental Example 1

[0041] 1. Test materials

[0042] Select well-growing caladium potted plants (such as Figure 1As shown in the figure, first use Nongdile diluted 1000 times for root irrigation to eliminate the eggs in the substrate, and then use Guoguang Yinna diluted 2000 times for leaf spraying and sterilization. For dry treatment, select the leaves of one-year-old plants without diseases and pests as explants after the water has completely dried up.

[0043] 2. Treatment of explants

[0044] For the newly cut Caladium bicolor, take its leaves, place them in a dish of dishwashing liquid water, gently brush them clean with a brush, and rinse them under running tap water for 0.5 h. Rinse the leaves 3 times with sterile water, disinfect them with 75% alcohol for 10 s, then rinse them 3 times with sterile water, disinfect them with 0.1% mercuric chloride for 8 min, and finally rinse them 6 times with sterile water (shake them manually for 1 min each time during the above sterile water rinsing). Blot the surface moisture with sterile filter paper. On the ultra-clean workbench, use a surgical blade to cut the disinfected leaves into 1-cm segments, and cut off the tissue at the cut surface (as Figure 2 shown).

[0045] 3. Callus induction

[0046] Using MS as the basic medium, a total of 4 kinds of media were designed for the induction of callus from Caladium bicolor explants, namely C1: MS + 0.5 mg / L 6-BA; C2: MS + 1.0 mg / L 6-BA + 0.1 mg / L IBA; C3: MS + 2.0 mg / L 6-BA + 0.1 mg / L IBA; C4: MS + 3.0 mg / L 6-BA + 1 mg / L TDZ + 0.1 mg / L IBA; the pH value of all media was 5.8, and 30 g / L of sucrose and 5 g / L of carrageenan were added to each formula. A total of 80 bottles were inoculated, with 3 - 4 leaves inoculated in each bottle. After inoculation, place them in the culture room for cultivation, with a light intensity of 45 μmol·m -2 ·s -1 , dark culture (without light) for the first week, and light culture after one week, with a light time of 12 h / d and a culture temperature of 25 °C. After 30 days of cultivation, start to compare the callus induction of different formulas (as Figure 3 shown).

[0047] Experimental results: As shown in Table 1.

[0048] Table 1 Formation of induced callus

[0049]

[0050] As can be seen from Table 1, in ordinary tissue culture rapid propagation, 6BA hormone is used for proliferation and induction. 6BA can induce callus in Caladium bicolor explants, but the efficiency of using TDZ hormone plus 6BA to jointly induce callus in explants is higher.

[0051] 4. Proliferation culture by different cutting methods

[0052] (1) Ordinary method

[0053] Cut callus on the induction material, cut the callus into small masses with a diameter of 0.2 cm, and culture them in a proliferation medium (MS basal medium + 30 g / L sucrose + 5 g / L carrageenan + 3.0 mg / L 6-BA + 1.0 mg / L TDZ + 0.1 mg / L IBA) under light conditions. The light intensity is 45 μmol·m -2 ·s -1 , the light time is 12 h / d, the temperature is 25 °C, and culture for 20 days. The shoots in each bottle can be subcut into three bottles of small shoots, and the subculture coefficient is about 3. As Figure 4 shown.

[0054] (2) Garlic paste smashing method

[0055] Cut callus on the induction material, cut it into three small masses with a diameter of 0.2 cm, and place them in a proliferation medium (MS basal medium + 30 g / L sucrose + 5 g / L carrageenan + 3.0 mg / L 6-BA + 1.0 mg / L TDZ + 0.1 mg / L IBA). The light intensity is 45 μmol·m -2 ·s -1 , the light time is 12 h / d, the temperature is 25 °C, culture in the culture room for 20 days until the small masses grow into masses with a size of 0.5 cm. Use forceps to take out the masses and disperse them by the garlic paste smashing method in a blender. After dispersion, use forceps to take them out and place them in a bottle. Put about 10 small shoots or fine powder in each bottle and evenly smear them on the surface of the proliferation medium (MS basal medium + 30 g / L sucrose + 5 g / L carrageenan + 3.0 mg / L 6-BA + 1.0 mg / L TDZ + 0.1 mg / L IBA). Each bottle can be subcultured to 30 bottles after smashing. The propagation coefficient is about 30. As Figure 5 shown.

[0056] Experimental results: As shown in Table 2.

[0057] Table 2 Comparison of proliferation coefficients of different cutting methods

[0058]

[0059] From Table 2 and Figure 4 、 Figure 5 it can be seen that for the ordinary cutting method, 1 mass becomes 3 masses. However, according to the characteristics of the callus of Caladium bicolor, it has dense growth points. According to the growth point characteristics of this variety, the garlic paste smashing method is used to smash the callus, and the cutting coefficient becomes more than 30, which is 10 times higher than that of ordinary cutting, greatly saving manpower and significantly reducing the propagation cost of tissue culture seedlings.

[0060] As can be seen from the above examples and experimental examples, the method of the present invention can significantly improve the callus induction efficiency and proliferation coefficient of Caladium bicolor, and greatly reduce the reproduction cost of Caladium bicolor tissue culture seedlings.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A tissue culture rapid propagation method for caladium plants, characterized in that: The following steps are involved: 1) using stems or leaves of caladium plants as explants, sterilizing them, and inoculating them into callus induction medium for culture to obtain callus; 2) The callus tissue is dispersed by mashing garlic and then inoculated into a proliferation culture medium for proliferation culture.

2. The tissue culture rapid propagation method according to claim 1, characterized in that: Step 1) The callus induction medium is based on MS and further comprises the following components in concentrations: 2-4 mg / L 6-BA, 0.5-1.5 mg / L TDZ, 0.05-0.15 mg / L IBA, 25-35 g / L sucrose and 3-7 g / L carrageenan.

3. The tissue culture rapid propagation method according to claim 1, characterized in that: Step 1) The culture temperature is 23-27°C, the culture method is dark culture and light culture, the dark culture time is 5-9 days, and the light intensity of the light culture is 40-50 μmol·m -2 ·s -1 The illumination time of the light culture is 10 to 14 h / d, and the time of the light culture is 21 to 25 days.

4. The tissue culture rapid propagation method according to claim 1, characterized in that: Step 2) The proliferation medium is based on MS and further comprises the following components at the following concentrations: 2-4 mg / L 6-BA, 0.5-1.5 mg / L TDZ, 0.05-0.15 mg / L IBA, 25-35 g / L sucrose and 3-7 g / L carrageenan.

5. The tissue culture rapid propagation method according to claim 1, characterized in that: Step 2) The proliferation culture is carried out by light culture, and the light intensity of the proliferation culture is 40-50 μmol·m -2 ·s -1 The illumination time of the proliferation culture is 10 to 14 h / d, the temperature of the proliferation culture is 23 to 27° C., and the time of the proliferation culture is 18 to 22 days.

6. The tissue culture rapid propagation method according to claim 1, characterized in that: The length of the explant is 0.8-1.2 cm.

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