Method for inducing anthurium callus and synchronously differentiating to generate adventitious buds

Abstract

The invention relates to a method for inducing anthurium callus and synchronously differentiating to generate adventitious buds, which comprises the following steps: 1) selecting explants; 2) induced differentiation culture; and (3) subculture multiplication culture: inoculating adventitious buds generated by induced differentiation into a multiplication culture medium for culture to obtain single seedlings with good growth vigor, and controlling the culture time to be 38-42 days. According to the method, the process from callus induction to differentiation seedling formation of the anthurium explant can be completed in the same culture medium, the stages of callus proliferation culture, differentiation culture and the like are not needed, the adventitious buds can be directly obtained from the explant only by culturing for about 40 days, the culture period is greatly shortened, the factory production cost is effectively reduced, and the method is suitable for industrial production. The economic benefit and the social benefit are improved. According to the method, the key technical bottleneck from explant induced dedifferentiation to redifferentiation in anthurium andraeanum tissue culture is solved, the anthurium andraeanum tissue culture period is greatly shortened, and the anthurium andraeanum tissue culture production cost is greatly reduced.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for inducing anthurium andraeanum callus and synchronously differentiating and producing adventitious buds. Background Art

[0002] Anthurium, also known as Anthurium and Anthurium, belongs to the genus Anthurium in the Araceae family. It prefers warmth and dislikes cold, prefers humidity and dislikes drought, prefers shade and dislikes direct sunlight. Its flowers are unique, with a spike-shaped inflorescence that is vibrant and rich in color. Each flower blooms for a long time, with a wide range of color variations. From the time the bracts open to the time the flowers wither and fall, the inflorescence undergoes a series of color changes, from beige to creamy white, finally turning green, and finally yellow before withering. Its leaf-shaped bracts, commonly found in colors such as red, pink, and white, are highly ornamental.

[0003] Anthurium andraeanum has a slow-growing fleshy root system and a low reproduction rate, making conventional division propagation difficult to meet the needs of factory production. Therefore, it is necessary to establish a comprehensive tissue culture and rapid propagation system for anthurium andraeanum, which can not only solve the problem of its slow reproduction rate, but also produce large quantities of anthurium andraeanum seedlings with consistent growth and high quality to meet market demand. The current plant tissue culture process generally includes: explant preparation stage, callus induction stage, callus proliferation culture stage, callus differentiation and adventitious bud production culture stage, subculture stage, rooting culture stage, seedling hardening and transplanting stage, etc. The culture cycle for each stage is approximately 30-60 days. The long culture cycle, slow growth rate, and high cost have seriously restricted the factory production of anthurium andraeanum tissue culture. Summary of the Invention

[0004] The invention provides a method for inducing anthurium callus and synchronously differentiating it to produce adventitious buds, so as to overcome the shortcomings of the existing anthurium tissue culture, such as long cycle, slow growth rate and high cost, which seriously restrict the factory production of anthurium tissue culture.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A method for inducing callus tissue of anthurium andraeanum and synchronously differentiating it to produce adventitious buds comprises the following steps: 1) selecting an explant, selecting an anthurium andraeanum plant that is robust and free of pests and diseases, collecting young leaves as explants, and then disinfecting the explants; 2) inducing differentiation culture, inoculating the disinfected young leaves into an inducing differentiation medium for culturing to obtain adventitious buds, with the culturing time being controlled to be 38-42 days; and 3) subculture proliferation culture, inoculating the adventitious buds produced by the induced differentiation into a proliferation medium for culturing to obtain well-growing single seedlings, with the culturing time being controlled to be 38-42 days.

[0007] Furthermore, the disinfection method in step 1) is as follows: the explants are rinsed with clean water under running water for 30 minutes to remove dust and other impurities on the surface; after being disinfected with 75% ethanol for 25-35 seconds, they are immediately placed in 0.1% HgCl2 for disinfection for 8-12 minutes, and then washed with sterile water 5 times; finally, the disinfected explants are cut into small pieces of leaves of 2 cm×2 cm in size and spread flat on the induction medium.

[0008] Furthermore, in step 2), the culture temperature is controlled at 22° C.-26° C., the illumination time is controlled at 10-14 h / d, and the illumination intensity is controlled at 2000-2500 lx.

[0009] By adopting the above technical solution, the present invention has the following beneficial effects: the method for inducing callus tissue of anthurium andraeanum and simultaneously differentiating it to produce adventitious buds enables the process of inducing callus tissue and differentiating it into seedlings from anthurium andraeanum explants to be completed in the same culture medium (i.e., induction and differentiation medium), eliminating the need for callus proliferation culture and differentiation culture stages. Adventitious buds can be directly obtained from the explants in just 40 days of culture, significantly shortening the culture cycle, effectively reducing factory production costs, and improving economic and social benefits. This method directly differentiates adventitious buds from callus tissue generated from the explant, reducing the number of callus proliferation cultures and thus significantly reducing the risk of seedling degeneration or mutation. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is a diagram showing the growth effect of adventitious buds produced by callus tissue after 40 days of culture using the method of the present invention. DETAILED DESCRIPTION

[0011] Specific embodiments of the present invention will be described below with reference to the accompanying drawings.

[0012] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0013] A method for inducing callus and synchronously differentiating adventitious buds in anthurium andraeanum comprises the following steps: 1) selecting an explant: selecting a robust, pest-free anthurium andraeanum plant, collecting young leaves as the explant, and then disinfecting the explant; 2) rinsing the explant with clean water under running water for 30 minutes to remove surface dust and other impurities; 3) disinfecting the explant with 75% ethanol for 25-35 seconds, immediately placing it in 0.1% HgCl2 for 8-12 minutes, and then rinsing it five times with sterile water; 4) cutting the disinfected explant into 2 cm x 2 cm leaf pieces, and paving the pieces flatly in an induction medium. The anthurium andraeanum plant is a robust, pest-free, new anthurium cultivar 'Flamingo' grown in the greenhouse of the Fujian Forestry Science and Technology Experimental Center.

[0014] In addition, before the small leaves are spread on the induction culture medium, they can also be subjected to auxiliary germination treatment. The auxiliary germination treatment method is: the small leaves are treated with a uniform electric field. Specifically, the small leaves are placed in an induction container, and then the small leaves in the induction container are induced by a 100kv-200kv / m uniform electric field (optimal 150kv / m) emitted by a high-voltage electrostatic generator. The induction treatment time is controlled within 1-2 minutes. Studies have shown that the induction treatment time of the small leaves in the high-voltage electrostatic field is within 1-2 minutes. As the treatment time increases, the activity of the dehydrogenase of the small leaves increases significantly. The treatment effect reaches the best when the treatment time is 1.5 minutes. After more than 2 minutes, as the treatment time continues to increase, the treatment effect gradually weakens.

[0015] 2) Induced differentiation culture: inoculate the sterilized young leaves into the induction differentiation medium for culture to obtain adventitious buds. The culture temperature is controlled at 22°C-26°C, the light duration is controlled at 10-14h / d, the light intensity is 2000-2500lx, and the culture time is controlled at 38-42 days. The induction differentiation medium is: 1 / 2MS + 1.5mg·L -1 6-BA+0.3mg·L -1 NAA.

[0016] The screening method of the differentiation induction medium is as follows: using different basic culture media (i.e., MS, 1 / 2MS, 1 / 3MS) and different hormone combinations (6-BA, NAA), adopting a 3-factor 3-level L9 (34) orthogonal design, 9 treatments, using leaves (i.e., young leaves) as explants, inoculating them into different differentiation induction culture media, observing and counting the growth conditions of different explants, including the formation and status of callus tissue, etc.; after 60 days of inoculation, counting the callus differentiation rates of explants in different combinations, and screening out the most suitable differentiation induction culture medium.

[0017] Table 1 Experimental design of explant induction medium

[0018]

[0019] As can be seen from Table 1, the improved induction differentiation medium used in this experiment can promote the differentiation of anthurium callus and the induction of adventitious buds in the same medium, and the time for forming adventitious buds is only 40 days.

[0020] Table 2 Range analysis of different culture media and hormone concentrations on the induction of anthurium explants

[0021]

[0022]

[0023] From the results of the three-factor, three-level orthogonal experiment on callus induction and differentiation of anthurium andraeanum using different culture media and hormone concentrations of plant growth regulators in Table 2, it can be seen that the induction and differentiation rates of explants in each treatment were different, among which the average induction and differentiation rate of treatment 2 was the highest, at 82.60%. The R values ​​of each factor were also different, indicating that the three factors had different degrees of influence on the induction and differentiation of anthurium andraeanum. Among them, the effect of 6-BA (B) was significantly higher than that of the other two factors, while the influence of culture medium (A) and NAA (C) was relatively close. The results of the comprehensive K value showed that the optimal combination was A2B2C2, that is, 1 / 2MS+6-BA (1.5 mg·L -1 )+NAA(0.3mg·L -1 ), the induction differentiation effect is better.

[0024] 3) Subculture proliferation culture: inoculate the adventitious buds produced by induced differentiation into a proliferation medium for cultivation to obtain well-growing single seedlings. The culture time is controlled to be 38-42 days (e.g., 40 days). The proliferation medium is: MS + 0.6 mg·L -1 6-BA+0.2mg·L -1 IBA.

[0025] The screening method of the proliferation culture medium is as follows: using MS culture medium as the base culture medium, different types and concentrations of exogenous plant growth regulators are added, among which 6-BA (0.4 mg·L -1 , 0.6mg·L -1 , 0.8mg·L -1 ) and IBA (0 mg·L -1 , 0.2mg·L -1 ), with a total of 6 different combinations. The induced adventitious buds were inoculated into different proliferation media and cultured under different culture conditions. The effects of different exogenous plant growth regulator combinations on the adventitious bud status, proliferation coefficient, etc. during the proliferation culture process were compared to screen for the optimal proliferation culture.

[0026] Table 3 Effects of different hormone levels on proliferation and growth of Anthurium andraeanum

[0027]

[0028]

[0029] As shown in Table 3, different hormone levels have a significant effect on the proliferation and growth of anthurium. Adding 6-BA and IBA at the same time can better promote the proliferation and growth of anthurium and increase the proliferation coefficient. As the concentration increases, the proliferation coefficient also increases accordingly. However, too high a concentration of 6-BA will also affect the growth of anthurium, resulting in weak buds, yellow leaves, curling and deformation. The experiment showed that adding 0.6 mg·L-1 6-BA and 0.2 mg·L -1 IBA can effectively promote the proliferation and growth of anthurium andraeanum, with a proliferation coefficient as high as 3.44.

[0030] The above is only a specific implementation of the present invention, but the design concept of the present invention is not limited to this. Any non-substantial changes to the present invention using this concept shall be deemed as an infringement of the protection scope of the present invention.

Claims

1. A method for inducing callus and synchronously differentiating it to produce adventitious buds in Anthurium andraeanum, characterized by: The method comprises the following steps: 1) selection of explants, selecting robust and pest-free anthurium plants, collecting young leaves as explants, and then disinfecting the explants; 2) induction differentiation culture, inoculating the disinfected young leaves into an induction differentiation medium for cultivation to obtain adventitious buds, with the cultivation time controlled at 38-42 days; and 3) subculture proliferation culture, inoculating the adventitious buds produced by induced differentiation into a proliferation medium for cultivation to obtain well-growing single seedlings, with the cultivation time controlled at 38-42 days.

2. The method for inducing callus and synchronously differentiating adventitious buds from Anthurium andraeanum according to claim 1, characterized in that: The disinfection method in step 1) is as follows: the explants are rinsed with clean water under running water for 30 minutes to remove dust and other impurities on the surface; after disinfection with 75% ethanol for 25-35 seconds, they are immediately placed in 0.1% HgCl2 for disinfection for 8-12 minutes, and then rinsed with sterile water five times; finally, the disinfected explants are cut into small leaves of 2 cm×2 cm in size and spread flat on the induction medium.

3. The method for inducing callus and synchronously differentiating adventitious buds of Anthurium andraeanum according to claim 1, characterized in that: In step 2), the culture temperature is controlled at 22° C.-26° C., the illumination time is controlled at 10-14 h / d, and the illumination intensity is controlled at 2000-2500 lx.

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