Method for improving growth speed and survival rate of Guangdong rohdea japonica plant tissue culture and increasing red color of leaves

Abstract

The invention provides a method for improving tissue culture growth speed and survival rate of Guangdong rohdea japonica plants and increasing red color of leaves, which comprises the following steps: selecting stems and / or tender leaves of the Guangdong rohdea japonica plants as explants, sterilizing, inoculating into an axillary bud induction culture medium, and performing primary culture under proper conditions; inoculating adventitious buds obtained by primary culture into a proliferation culture medium, and carrying out subculture under proper conditions; selecting a single bud seedling with the height of 4-5cm from the multiple shoots subjected to subculture, inoculating the single bud seedling into a rooting culture medium, and carrying out rooting culture, so as to obtain a tissue culture seedling with a complete root and bud structure. According to the method, nano-zinc oxide with a certain concentration is respectively added into an axillary bud induction culture medium and a proliferation culture medium and then is in synergistic interaction with other nutritional ingredients in the culture medium, so that rapid germination is facilitated, the growth rate and the robustness of plants are improved, and the nano-zinc oxide participates in plant pigment synthesis; the development and inheritance of the red character in the Guangdong rohdea japonica leaves are facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant tissue culture, and specifically relates to a method for improving the growth rate and survival rate of tissue culture of Aglaonema commutatum plants and increasing the red color of leaves. Background Art

[0002] Aglaonema commutatum belongs to the perennial evergreen herb of the genus Aglaonema in the Araceae family, also known as Liang Sicao. It grows in areas with an altitude of 500 to 1700 meters, mostly in dense forests.

[0003] In terms of growth habits, Aglaonema commutatum prefers high-temperature and high-humidity climates and is suitable for growing in an environment with high air humidity and relatively high temperature. It has strong shade tolerance and poor tolerance to direct sunlight, which is likely to cause leaf burns, resulting in phenomena such as fading, yellowing, and even withering. It is suitable for growing in loose, fertile, and slightly acidic soil, which is conducive to root respiration and nutrient absorption, providing sufficient power for the continuous growth of the plant. Moreover, due to its strong adaptability, it can be placed indoors for a long time, with strong plant growth and a low incidence of pests and diseases, and is widely used in the field of indoor green plant maintenance.

[0004] In terms of application value, Aglaonema commutatum has certain medicinal value, and the whole plant can be used as medicine. According to the "Record of Medicinal Herbs Collected in the Lingnan Region", its leaves and lean meat are used to make soup, which has a significant effect on treating diseases such as hot blood, hemoptysis, heat accumulation in the large intestine, and rectal prolapse in children; its stems and leaves are mashed with sugar tablets and applied externally, which has a certain role in the adjuvant treatment of rabies bites. In addition, Aglaonema commutatum also has high ornamental value. Its plant type is compact and dignified, and its leaves are evergreen and shiny all year round, making it an excellent indoor foliage plant. The growth process of its roots can be observed by stem cuttings in indoor glassware. After pruning and matching, it can also be used as cut flowers to add natural beauty and artistic atmosphere to the indoor environment.

[0005] It is worth mentioning that some varieties of Aglaonema commutatum have unique red-mottled leaves, which significantly enhance their ornamental value. According to our research, the red color of Aglaonema commutatum leaves is mainly due to the synthesis and accumulation of pigments in the plant. At certain specific growth stages, through the combined action of a series of environmental factors such as temperature fluctuations, adjustment of light duration, and nutrient supply, the relevant physiological mechanisms are activated, promoting an increase in the content of pigments such as anthocyanins, and thus causing the leaves to change from the common green color to the mottled leaf color of coexisting red and green. In the indoor environment, when Aglaonema commutatum with red-mottled leaves is placed together with ordinary green plants, obvious color differences can be formed, injecting vivid visual interest into the monotonous indoor space and adding vitality.

[0006] At present, the conventional propagation methods of Aglaonema modestum are cutting and division. We studied the culture method for inducing tissue culture buds. The stem segments of Aglaonema modestum were placed in a common bud induction medium and cultured under appropriate environmental conditions. However, it was found that the budding time period of the stem segments of Aglaonema modestum requires 45 - 50 days to grow into a state where the next step of bud proliferation can be carried out, which takes a long time. Moreover, since Aglaonema modestum mainly has a greenish - blue leaf color, the red leaf color is less and it is prone to problems such as fading of the red leaf color and reduction of the red degree after long - term propagation, making it difficult to stably cultivate.

[0007] In summary, how to rapidly and massively propagate Aglaonema modestum through plant tissue culture technology, which can not only improve the growth rate and survival rate of Aglaonema modestum in tissue culture, but also enhance the red leaf color state, is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for improving the growth rate, survival rate of Aglaonema modestum in tissue culture, and increasing the red color of the leaves. Through appropriate medium design and optimization of the culture environment, it not only improves the growth rate of Aglaonema modestum, the germination rate, sprout length and quality during the bud induction process, but also increases the proportion of the red color of the leaves of Aglaonema modestum.

[0009] To achieve the above - mentioned purpose, the present invention provides a method for improving the growth rate and survival rate of Aglaonema modestum in tissue culture and increasing the red color of the leaves, including the following steps:

[0010] Select the stem segments and / or young leaves of Aglaonema modestum plants as explants. After disinfection treatment, inoculate them into the axillary bud induction medium and carry out primary culture under suitable conditions;

[0011] Inoculate the adventitious buds obtained from the primary culture into the proliferation medium and carry out sub - culture under suitable conditions;

[0012] Among the clustered buds after sub - culture, select single sprout seedlings with a height of 4 - 5 cm and inoculate them into the rooting medium for rooting culture, and then tissue - cultured seedlings with complete root and bud structures can be obtained.

[0013] In a preferred embodiment, the stem segment explant is the tender part with a length of 0.5 - 1 cm; the young leaf explant is the newly unfolded complete young leaf.

[0014] In a preferred embodiment, the disinfection treatment includes the following steps:

[0015] Place the explants under running water and gently rinse for 1 - 2 minutes to remove surface dust and impurities;

[0016] Soak the rinsed explants in a mixed solution containing 100 mg / L penicillin and 100 mg / L streptomycin for 30 minutes for preliminary antibacterial inhibition.

[0017] On a clean bench, soak the antibacterial-inhibited explants in 75% alcohol solution for 20 - 40 seconds and quickly take them out; then put the explants into a 0.1% mercuric chloride solution by mass concentration, add 2 - 3 drops of Tween - 80, soak for 8 - 10 minutes, gently shake constantly during this period, and finally rinse 3 - 4 times with sterile water and air dry.

[0018] In a preferred embodiment, the axillary bud induction medium is based on the modified MS medium, supplemented with 3 mg / L of 6 - benzylaminopurine, 0.5 mg / L of phenylthiadiazolylurea, 0.1 mg / L of indolebutyric acid, 50 mg / L of proline, 100 mg / L of hydrolyzed casein, 10 - 30 mg / L of nano - zinc oxide, and distilled water is added to make up 1 L; adjust the pH of the medium to 5.8 - 6.2.

[0019] In a preferred embodiment, the modified MS medium means that the macro - elements in the traditional MS medium are halved.

[0020] In a preferred embodiment, the primary culture conditions include: in an environment of 25 ± 2 °C, using a light source of 3800 - 4200 Lux, with a light cycle of 18 h light / 6 h darkness, and culturing for 15 days.

[0021] In a preferred embodiment, the proliferation medium is based on the MS medium, supplemented with 3.0 mg / L of 6 - benzylaminopurine, 0.05 mg / L of naphthylacetic acid, 0.5 mg / L of gibberellin, 100 ml / L of coconut milk, 35 g / L of sucrose, 7 g / L of agar, 10 - 30 mg / L of nano - zinc oxide, and distilled water is added to make up 1 L; adjust the pH of the medium to 5.8 - 6.2.

[0022] In a preferred embodiment, the sub - culture conditions include: in an environment of 26 ± 2 °C, using a light source of 4200 - 4700 Lux, and culturing according to a light cycle of 16 h light / 8 h darkness. Among them, sub - culture is carried out once every 20 days, and the sub - culture is carried out 2 - 4 times.

[0023] In a preferred embodiment, the rooting medium is based on 1 / 3 of the MS medium, supplemented with 0.3 mg / L of indole - 3 - butyric acid, 0.2 mg / L of rooting powder ABT, 100 mg / L of inositol, 50 mg / L of vitamin C, 20 g / L of glucose, and distilled water is added to make up 1 L; adjust the pH of the medium to 5.8 - 6.2.

[0024] In a preferred embodiment, the rooting culture conditions include: culturing for 30 days at 25 ± 1 °C under a light source of 3200 - 3700 Lux with a light cycle of 14 h light / 10 h darkness.

[0025] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0026] 1. In the present invention, young stem segments of 0.5 - 1 cm and newly developed complete young leaves are selected as explants, and the purpose is that: young stem segments have the strongest meristematic ability, and new leaf cells have high activity, both of which are beneficial to subsequent induction culture.

[0027] 2. In the present invention, by adding a certain concentration of nano - zinc oxide to the axillary bud induction medium and the proliferation medium respectively, and synergistically enhancing the effect with other nutrient components in the medium, it not only helps to germinate quickly, improve the plant growth rate and robustness, but also participates in the synthesis of plant pigments, which is beneficial to the appearance and inheritance of the red trait in the leaves of Aglaonema modestum. Specific Embodiments

[0028] In order to enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below in conjunction with specific embodiments, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0029] Unless otherwise specified, the technical means used in the present invention are conventional means well - known to those skilled in the art. All kinds of raw materials, reagents, instruments and equipment used in the present invention can be obtained through the market or can be prepared by existing methods. The reagents used in the present invention are of analytical grade unless otherwise specified. The Aglaonema modestum tissue culture materials used in the present invention are from healthy Aglaonema modestum plants of Hainan Dongfang Shangcai Modern Agriculture Co., Ltd.

[0030] Examples

[0031] A method for improving the tissue culture growth rate, survival rate and leaf redness of Aglaonema modestum plants, comprising the following steps:

[0032] Select young stem segments of Aglaonema modestum with a length of 0.5 - 1 cm as explants. After disinfection treatment, inoculate them into the axillary bud induction medium, with 3 shoot tips in each bottle, inoculate 5 bottles, and repeat the experiment 3 times. Under the environment of 25 °C, using a light source of 4000 Lux, according to the light cycle of 18 h light / 6 h darkness, conduct the primary culture for 15 days. The axillary bud induction medium used is based on the modified MS medium (halve the macroelements in the traditional MS medium), supplemented with 3 mg / L of 6-benzylaminopurine, 0.5 mg / L of thidiazuron, 0.1 mg / L of indolebutyric acid, 50 mg / L of proline, 100 mg / L of hydrolyzed casein, 10 - 30 mg / L of nano-zinc oxide, and add distilled water to 1 L; adjust the pH of the medium to 5.8 - 6.2. Among them, the specific dosage of nano-zinc oxide is shown in Table 1.

[0033] Inoculate the adventitious buds obtained from the primary culture into the proliferation medium. Under the environment of 26 °C, use a light source of 4500 Lux, and conduct subculture according to the light cycle of 16 h light / 8 h darkness. Among them, subculture is carried out once every 20 days, and a total of 3 subcultures are carried out. The proliferation medium used is based on the MS medium, supplemented with 3.0 mg / L of 6-benzylaminopurine, 0.05 mg / L of naphthaleneacetic acid, 0.5 mg / L of gibberellin, 100 ml / L of coconut milk, 35 g / L of sucrose, 7 g / L of agar, 10 - 30 mg / L of nano-zinc oxide, and add distilled water to 1 L; adjust the pH of the medium to 5.8 - 6.2. Among them, the specific dosage of nano-zinc oxide is shown in Table 1.

[0034] Among the clustered buds after subculture, select single bud seedlings with a height of 4 - 5 cm and inoculate them into the rooting medium. Under the environment of 25 ± 1 °C, use a light source of 3500 Lux, according to the light cycle of 14 h light / 10 h darkness, conduct rooting culture for 30 days, and then tissue culture seedlings with complete root and bud structures can be obtained. The rooting medium used is based on 1 / 3 of the MS medium, supplemented with 0.3 mg / L of indole-3-butyric acid, 0.2 mg / L of rooting powder ABT, 100 mg / L of inositol, 50 mg / L of vitamin C, 20 g / L of glucose, and add distilled water to 1 L; adjust the pH of the medium to 5.8 - 6.2.

[0035] Table 1 Dosage of nano-zinc oxide in different media (mg / L)

[0036] Example Axillary bud induction medium Proliferation medium Example 1 0 0 Example 2 0 10 Example 3 0 20 Example 4 0 30 Example 5 10 0 Example 6 10 10 Example 7 10 20 Example 8 10 30 Example 9 20 0 Example 10 20 10 Example 11 20 20 Example 12 20 30 Example 13 30 0 Example 14 30 10 Example 15 30 20 Example 16 30 30

[0037] Data processing and results: The growth cycle and multiplication coefficient of tissue-cultured seedlings were statistically analyzed. The formula for calculating the multiplication coefficient is: Multiplication coefficient = the number of newly generated plants after subculture / the number of explants at inoculation. The plant height, leaf width, and length data of the tissue-cultured seedlings obtained in each example were measured and the average values were taken. The red parts of the leaves of the tissue-cultured seedlings in each example were cut out, and the proportion of the red patch leaf area was calculated by the leaf shape paper weighing method. The obtained data are shown in Table 2.

[0038] Table 2

[0039] Example Proliferation coefficient Plant height (cm) Leaf width (cm) Leaf length (cm) Proportion of red on leaf surface Example 1 2.69 2.36 1.65 2.33 15.55％ Example 2 3.03 2.57 1.72 2.52 20.48％ Example 3 3.16 2.63 1.85 2.60 26.20％ Example 4 2.81 2.41 1.70 2.44 18.94％ Example 5 3.02 2.59 1.76 2.57 22.13％ Example 6 4.74 3.18 2.06 2.93 40.85％ Example 7 5.83 3.63 2.19 3.28 46.86％ Example 8 4.55 3.00 2.05 2.82 39.41％ Example 9 3.14 2.67 1.89 2.79 28.49％ Example 10 6.85 3.71 2.25 3.57 50.34％ Example 11 8.14 3.83 2.27 3.77 51.97％ Example 12 5.62 3.52 2.12 3.25 44.95％ Example 13 2.99 2.55 1.65 2.46 20.74％ Example 14 3.79 2.91 1.98 2.79 37.82％ Example 15 4.81 3.23 2.10 3.22 41.61％ Example 16 3.59 2.85 1.95 2.70 32.69％

[0040] As can be seen from Table 2, by adding an appropriate amount of nano-zinc oxide to the axillary bud induction medium and proliferation medium, the growth rate and plant quality of Aglaonema modestum tissue-cultured seedlings can be effectively improved. Moreover, compared with the traditional tissue-culture cycle of 8 - 10 weeks, the method of the present invention can shorten the culture cycle to 2 - 3 weeks, greatly improving the cultivation efficiency. In addition, the present invention promotes the stable inheritance of leaf red mottling by promoting plant photosynthesis, adjusting plant hormone levels, improving nutrient absorption and antioxidant capacity, etc., avoiding problems such as leaf color fading and reduction of red degree, thereby cultivating high-quality red and green mottled Aglaonema modestum.

[0041] We also found that the specific nano-zinc oxide concentration in the axillary bud induction medium and proliferation medium has an important influence on the experimental effect. Exploring the mechanism, the reason is that if the concentration is too high (greater than 30 mg / L), it will have certain side effects on Aglaonema cells, which will instead affect the normal metabolism and physiological functions of cells. For example, high-concentration nano-zinc oxide will damage the chloroplast structure and inhibit the activity of photosynthesis-related enzymes, thereby reducing the photosynthesis efficiency, making the leaves unable to obtain enough energy and substances to synthesize pigments, resulting in the fading of leaf red color. At the same time, high-concentration nano-zinc oxide will also have an antagonistic effect with other nutrient elements, affecting the absorption, transportation and utilization of other essential elements by Aglaonema modestum or inhibiting the synthesis and signal transduction of plant hormones, breaking the hormone balance in plants. If the concentration is too low (less than 10 mg / L), the functions of promoting photosynthesis, regulating hormone levels, improving nutrient absorption and enhancing antioxidant capacity cannot be fully exerted, making the pigment synthesis and metabolism process unable to proceed fully, and the leaf red mottling cannot be effectively improved. Therefore, in the axillary bud induction medium and proliferation medium of the present invention, the appropriate concentration of nano-zinc oxide is 10 - 30 mg / L.

[0042] The foregoing description of specific exemplary embodiments of the present invention is for purposes of illustration and exemplification. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many modifications and variations are possible in light of the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the invention and its practical applications, so that those skilled in the art can implement and utilize the various different exemplary embodiments of the invention, as well as various different selections and modifications. The scope of the present invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for improving the tissue culture efficiency of Dieffenbachia glabra plants and the red color of their leaves, characterized in that: The following steps are involved: Select stem segments and / or tender leaves of Dieffenbachia glabra as explants, sterilize them, inoculate them in axillary bud induction medium, and carry out primary culture under suitable conditions; The adventitious buds obtained from the primary culture are inoculated into a proliferation medium and subcultured under suitable conditions; From the clustered buds after subculture, single buds with a height of 4-5 cm are selected and inoculated into a rooting medium for rooting culture, thereby obtaining tissue culture seedlings with complete root and bud structures.

2. The method according to claim 1, characterized in that: The stem segment explant is a young and tender part with a length of 0.5-1 cm; the young leaf explant is a newly unfolded complete young leaf.

3. The method according to claim 1, characterized in that: The disinfection process comprises the following steps: Rinse the explants gently under running water for 1-2 minutes to remove surface dust and impurities; The washed explants were immersed in a mixed solution containing 100 mg / L penicillin and 100 mg / L streptomycin for 30 min for preliminary bacteriostasis; On a clean workbench, soak the antibacterial explants in a 75% alcohol solution for 20-40 seconds and quickly take them out; then put the explants in a 0.1% mercuric chloride solution, add 2-3 drops of Tween-80, soak for 8-10 minutes, shake gently during the process, and finally rinse with sterile water 3-4 times and dry.

4. The method according to claim 1, characterized in that: The axillary bud induction culture medium is based on the improved MS culture medium, supplemented with 3 mg / L of 6-benzylaminopurine, 0.5 mg / L of phenylthiadiazolyl urea, 0.1 mg / L of indolebutyric acid, 50 mg / L of proline, 100 mg / L of hydrolyzed casein, 10-30 mg / L of nano zinc oxide, and distilled water to 1 L; the pH of the culture medium is adjusted to 5.8-6.

2.

5. The method according to claim 4, characterized in that: The improved MS medium refers to a medium in which the macroelements in the traditional MS medium are reduced by half.

6. The method according to claim 1, characterized in that: The primary culture conditions include: in an environment of 25±2° C., using a light source of 3800-4200 Lux, with a light cycle of 18 h light / 6 h dark, and culturing for 15 days.

7. The method according to claim 1, characterized in that: The proliferation culture medium is based on MS culture medium, supplemented with 3.0 mg / L 6-benzylaminopurine, 0.05 mg / L naphthaleneacetic acid, 0.5 mg / L gibberellin, 100 ml / L coconut juice, 35 g / L sucrose, 7 g / L agar, 10-30 mg / L nano zinc oxide, and distilled water to 1 L; the pH of the culture medium is adjusted to 5.8-6.

2.

8. The method according to claim 1, characterized in that: The subculture conditions include: culturing at 26±2°C, using a 4200-4700 Lux light source, and a light cycle of 16h light / 8h dark, wherein subculture is performed once every 20 days, and subculture is performed 2-4 times.

9. The method according to claim 1, characterized in that: The rooting medium is based on 1 / 3 of MS medium, supplemented with 0.3 mg / L indole-3-butyric acid, 0.2 mg / L rooting powder ABT, 100 mg / L inositol, 50 mg / L vitamin C, 20 g / L glucose, and distilled water to 1 L; the pH of the medium is adjusted to 5.8-6.

2.

10. The method according to claim 1, characterized in that: The rooting culture conditions include: in an environment of 25±1° C., using a light source of 3200-3700 Lux, with a light cycle of 14 hours of light / 10 hours of darkness, and culturing for 30 days.