A method for inducing callus and regenerating adventitious buds from Acer truncatum stem segments

Abstract

The invention discloses a method for inducing callus of Acer truncatum stem segments and regenerating adventitious buds, and relates to the technical field of plant rapid propagation. The invention inoculates Acer truncatum stem segments into callus induction medium for callus induction culture to obtain Acer truncatum callus; inoculates callus into adventitious bud differentiation medium for adventitious bud differentiation culture to obtain Acer truncatum callus differentiated with adventitious buds; cuts off the adventitious buds and transfers them to rooting medium for rooting culture until adventitious roots grow out of the base of the adventitious buds, thereby obtaining Acer truncatum regenerated plants. The results show that the combination of ①2,4-D and BA or ②IBA and BA in the callus induction medium has the best induction effect on the callus of Acer truncatum stem segments; CPPU is a key factor in inducing Acer truncatum callus to differentiate adventitious buds; and solves the problem of the difficulty of Acer truncatum callus differentiation in the prior art, which is of great significance for promoting the breeding and industrialization development of Acer truncatum.

Description

Technical Field

[0001] The invention belongs to the technical field of plant rapid propagation, and more specifically relates to a method for inducing callus tissue of Acer truncatum stem segments and regenerating adventitious buds. Background Art

[0002] Acer truncatum Acer truncatum Bunge belongs to the deciduous tree of the genus Acer in the family Aceraceae. It is a unique economic tree species in my country. It has many important values ​​such as ornamental, ecological and oil use. In particular, its oil value has attracted widespread attention from the pharmaceutical and food industries. As one of the few woody plant oil resources containing neuraminic acid (5%-6%), it has great development and application potential for repairing brain nerves, preventing and treating sequelae of cerebral stroke and Alzheimer's disease. It has been listed as a national key woody oil tree species and new resource food (Ministry of Health Announcement No. 9 of 2011), and has broad market prospects.

[0003] In recent years, the domestic Acer truncatum cultivation area and industrial system have grown rapidly. According to incomplete statistics, the domestic Acer truncatum planting area has reached more than 2 million mu. However, the current Acer truncatum reproduction and new variety cultivation mainly use traditional methods such as seed propagation and seedling breeding. The breeding efficiency is low, the cycle is long, and the seedling quality is unstable, which seriously restricts its industrial development. Therefore, establishing an efficient and stable Acer truncatum regeneration system is a bottleneck problem that needs to be solved urgently.

[0004] Tissue culture technology is an important means of rapid propagation of plant varieties and an important component of building a genetic transformation system. Among them, the use of explants to induce callus tissue and then regenerate adventitious buds is an important way for plant in vitro regeneration. There are few reports on tissue culture of Acer truncatum, and most of them are reports on direct rooting using stem segments with buds. There are few studies on its proliferation process, the reproduction efficiency is low, and this regeneration pathway is not suitable for genetic transformation. In addition, there are a few reports on the induction of callus tissue of Acer truncatum, but none of them have achieved callus differentiation, and there has been no report on the successful regeneration of callus tissue, which is a bottleneck problem restricting the in vitro regeneration of Acer truncatum. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a method for inducing callus tissue of Acer truncatum stem segments and regenerating adventitious buds, so as to solve the problem of difficult differentiation of callus tissue of Acer truncatum.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for inducing callus and regenerating adventitious buds from Acer truncatum stem segments, comprising:

[0008] 1) inoculating the stem segments of Acer truncatum into a callus induction medium for callus induction culture to obtain Acer truncatum callus;

[0009] 2) inoculating the callus into an adventitious bud differentiation medium for adventitious bud differentiation culture to obtain Acer truncatum callus that differentiates into adventitious buds;

[0010] 3) The adventitious buds are cut off and transferred to a rooting medium for rooting culture until adventitious roots grow out from the base of the adventitious buds, thereby obtaining regenerated plants of Acer truncatum.

[0011] The callus induction medium formula is NN69 medium + 30g / L sucrose + 6.5g / L agar + ①1mg / L2,4-D + 0.5mg / L BA; or ②0.5mg / L 2,4-D + 0.2mg / L BA; or ③0.2mg / L2,4-D + 0.5mg / L BA; or ④0.5mg / L NAA + 0.2mg / L BA; or ⑤0.2mg / L NAA + 0.5mg / LBA; or ⑥0.2mg / L IBA + 0.5mg / LBA; or ⑦0.5mg / L 2,4-D + 0.2mg / L CPPU; or ⑧0.5mg / L NAA + 0.2mg / L CPPU.

[0012] The callus induction culture conditions are as follows: a culture period of 20 days, dark culture throughout the process, and a culture temperature of 24±1°C.

[0013] The formula of the adventitious bud differentiation medium is NN69 medium + 30g / L sucrose + 6.5g / L agar + ①0.25mg / L CPPU; or ②0.5mg / L CPPU; or ③0.25mg / L TDZ; or ④0.5mg / L TDZ; or ⑤0.25mg / L CPPU + 0.25mg / L BA; or ⑥0.25mg / L CPPU + 0.25mg / L TDZ; or ⑦0.1mg / L NAA + 0.5mg / L CPPU; or ⑧0.1mg / L IBA + 0.5mg / L CPPU.

[0014] The adventitious bud differentiation culture conditions are as follows: a subculture period of 20 days, a culture photoperiod of 12 hours of light / 12 hours of darkness, and a culture temperature of 24±1°C.

[0015] The rooting medium formula is WPM medium+IBA 0.5 mg / L+30 g / L sucrose+AC 0.5 g / L+6.5 g / L agar.

[0016] The rooting culture conditions are as follows: the culture photoperiod is 12 hours of light / 12 hours of darkness, and the culture temperature is 24±1°C.

[0017] The method for inducing callus tissue and regenerating adventitious buds of Acer truncatum stem segments comprises the following specific steps:

[0018] 1) Inoculate the stem segments of Acer truncatum into callus induction medium, culture for 20 days, use dark culture throughout the process, and culture at 24±1°C to obtain Acer truncatum callus;

[0019] 2) Inoculating the Acer truncatum callus into an adventitious bud differentiation medium for differentiation culture, with a subculture period of 20 days, a culture photoperiod of 12 hours light / 12 hours dark, and a culture temperature of 24±1° C. to obtain the Acer truncatum callus that differentiates into adventitious buds;

[0020] 3) When the adventitious buds produced by callus differentiation grow to 1-2 cm, the adventitious buds are cut and inoculated into rooting medium for rooting culture. The culture light cycle is 12 h light / 12 h dark, and the culture temperature is 24±1°C. After about 1-2 weeks of culture, adventitious roots grow from the base of the adventitious buds, and the Acer truncatum regenerated plants are obtained.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1) The present invention inoculates Acer truncatum stem segments into callus induction culture medium for callus induction culture to obtain Acer truncatum callus. Different plant growth regulators were added to the callus induction culture medium, and the results showed that the callus induction rate of most of the treatments was above 80%, and the callus was mostly white, yellow-white or yellow, with a relatively dense texture, and some were nodular. Combined with the subsequent differentiation performance, medium No. 3 and No. 6 performed better, indicating that the combination of 2,4-D or IBA and BA had the best induction effect on the callus of Acer truncatum stem segments.

[0023] 2) The present invention inoculates callus into an adventitious bud differentiation medium for adventitious bud differentiation culture, and obtains Acer truncatum callus that differentiates adventitious buds. Different plant growth regulators were added to the adventitious bud differentiation medium, and the results showed that adventitious buds were differentiated on D1, D2 and D5 mediums, indicating that CPPU is a key factor in inducing adventitious bud differentiation of Acer truncatum callus. No adventitious bud differentiation was observed on D3, D4 and D6 mediums containing TDZ, indicating that TDZ is not suitable for inducing adventitious bud differentiation. No adventitious bud differentiation was observed on D7 and D8 mediums containing auxin, indicating that the addition of auxin may not be conducive to the differentiation of adventitious buds. In addition, callus that differentiated adventitious buds mainly came from callus induction medium No. 3 and No. 6, indicating that the early callus induction process has a great influence on the differentiation of adventitious buds.

[0024] 3) The present invention cuts off the adventitious buds and transfers them to a rooting medium for rooting culture until adventitious roots grow from the base of the adventitious buds, thereby obtaining a regenerated plant of Acer truncatum. Finally, the problem of the difficulty in differentiation of callus tissue of Acer truncatum in the prior art is solved, which is of great significance for promoting the breeding and industrial development of Acer truncatum. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a picture of the callus tissue of Acer truncatum stem segment (callus induction medium No. 3);

[0026] Figure 2 This is a picture of the callus tissue of Acer truncatum stem segment (callus induction medium No. 6);

[0027] Figure 3 This is a picture of adventitious buds differentiated from callus of Acer truncatum stem segment;

[0028] Figure 4 This is a graph of adventitious bud elongation;

[0029] Figure 5 It is a diagram of rooting of adventitious buds;

[0030] Figure 6 This is a diagram of transplanting rooted seedlings. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0032] The Acer truncatum used in the following examples comes from the maple seed base of Jiangsu Academy of Agricultural Sciences. In April 2023, young and tender branches of the year were selected, rinsed with running water for 5 minutes, and then placed in a clean workbench for disinfection, 75% ethanol disinfection for 30 seconds, 3% sodium hypochlorite disinfection for 10 minutes, rinsed with sterile water 4-5 times, and then the stem segments were placed on sterile filter paper to absorb moisture, and cut into stem segments of about 5 mm with a scalpel as explants.

[0033] Example 1

[0034] The stem segments of Acer truncatum were inoculated into callus induction medium, which were: ①NN69 medium + 30g / L sucrose + 6.5g / L agar + 1mg / L 2,4-D + 0.5mg / L BA; ②NN69 medium + 30g / L sucrose + 6.5g / L agar + 0.5mg / L2,4-D + 0.2mg / L BA; ③NN69 medium + 30g / L sucrose + 6.5g / L agar + 0.2mg / L 2,4-D + 0.5mg / L BA; ④NN69 medium + 30g / L sucrose + 6.5g / L agar + 0.5mg / L NAA + 0.2mg / L BA; ⑤NN69 medium + 30g / L sucrose + 6.5g / L agar + 0.2mg / L NAA + 0.5mg / L BA; ⑥NN69 medium + 30g / L sucrose + 6.5g / L agar + 0.2mg / L BA + 0.5mg / L BA; ⑦NN69 medium + 30g / L sucrose + 6.5g / L agar + 0.5mg / L 2,4-D + 0.2mg / L CPPU; ⑧NN69 medium + 30g / L sucrose + 6.5g / L agar + 0.5mg / L NAA + 0.2mg / L CPPU, the culture period is 20 days, the whole process is dark culture, the culture temperature is 24±1℃. The callus induction rate is counted.

[0035] The results are as follows Figure 1 , Figure 2 As shown, the Acer truncatum stem segments were inoculated on callus induction medium for about a week, and callus began to form at the cut ends.

[0036] The results are shown in Table 1. The callus induction rates of most treatments were above 80%. The callus induction rate of medium No. 1 was the highest, reaching 100%, and the lowest was 71% in medium No. 8. The callus induction rates of medium No. 2, No. 3 and No. 6 were all above 90%. The induction rates of medium No. 4, No. 5 and No. 8 using NAA were relatively low, indicating that the callus induction effect of NAA was poor. Callus tissue was mostly white, yellow-white or yellow, with a dense texture and some nodular. Combined with the subsequent differentiation performance, medium No. 3 and No. 6 performed better, indicating that the combination of ①2,4-D and BA or ②IBA and BA had the best induction effect on the callus of Acer truncatum stem segments.

[0037] Table 1 Effects of different plant growth regulators on callus induction of Acer truncatum

[0038]

[0039]

[0040] Example 2

[0041] After 20 days of callus induction, the callus was inoculated into adventitious bud differentiation medium for differentiation culture, which were: ①NN69 medium + 30g / L sucrose + 6.5g / L agar + 0.25mg / L CPPU; ②NN69 medium + 30g / L sucrose + 6.5g / L agar + 0.5mg / L CPPU; ③NN69 medium + 30g / L sucrose + 6.5g / L agar + 0.25mg / L TDZ; ④NN69 medium + 30g / L sucrose + 6.5g / L agar + 0.5mg / LTDZ; ⑤NN69 medium + 30g / L sucrose + 6.5g / L agar + 0.25mg / L CPPU + 0.25mg / L BA; ⑥NN69 medium + 30g / L sucrose + 6.5g / L agar + 0.25mg / L CPPU+0.25mg / L TDZ;⑦NN69 medium+30g / L sucrose+6.5g / L agar+0.1mg / L NAA+0.5mg / L CPPU;⑧NN69 medium+30g / L sucrose+6.5g / L agar+0.1mg / L IBA+0.5mg / L CPPU, subculture period is 20d, culture photoperiod is 12h light / 12h dark, culture temperature is 24±1℃. After 3 months of culture, the differentiation of adventitious buds was counted.

[0042] The results are as follows Figure 3 , Figure 4 As shown, the callus tissue of the Acer truncatum stem segment was inoculated on the adventitious bud differentiation medium and cultured under light. After a period of time, the callus tissue gradually turned green. After 3 months of culture, adventitious buds began to form on the surface of the callus tissue. With continued cultivation, the adventitious buds elongated and the leaves unfolded. After the adventitious bud stems elongated to 1-2 cm, they were cut off and transferred to the rooting medium for rooting.

[0043] The results are shown in Table 2. Among the treatments tested, adventitious buds were differentiated on D1, D2 and D5 mediums, indicating that CPPU is a key factor in inducing adventitious bud differentiation in Acer truncatum callus. No adventitious bud differentiation was observed on D3, D4 and D6 mediums containing TDZ, indicating that TDZ is not suitable for inducing adventitious bud differentiation. No adventitious bud differentiation was observed on D7 and D8 mediums containing auxin, indicating that the addition of auxin may be detrimental to the differentiation of adventitious buds. In addition, callus that differentiated adventitious buds mainly came from callus induction medium No. 3 and No. 6, indicating that the early callus induction process has a great influence on the differentiation of adventitious buds.

[0044] Table 2 Effects of different plant growth regulators on adventitious bud differentiation of Acer truncatum callus

[0045]

[0046] Example 3

[0047] When the adventitious buds generated by callus differentiation grow to 1-2 cm, the adventitious buds are cut and inoculated into rooting medium for rooting culture. The rooting medium uses WPM medium + IBA 0.5 mg / L + 30 g / L sucrose + AC 0.5 g / L + 6.5 g / L agar. The culture photoperiod is 12 h light / 12 h dark, and the culture temperature is 24 ± 1 ° C. After about 1-2 weeks of culture, adventitious roots grow from the base of the adventitious buds ( Figure 5 ) to obtain the regenerated plants of Acer truncatum. When the adventitious roots extend to more than 1 cm, take the seedlings out of the tissue culture bottle, wash the culture medium on the roots and transplant them ( Figure 6 ), the transplanting medium is peat: perlite: vermiculite = 3:1:1, and the humidity is enough to be able to be kneaded into a ball without falling apart. When transplanting, try to let the roots stretch naturally, and gently shake the medium to make it fit the roots, but it should not be pressed too tightly to avoid affecting ventilation. The transplanted seedlings are first hardened in an artificial climate room for 2 months (12h light / 12h dark, temperature 24±1℃, humidity 90%), and then moved to a greenhouse for cultivation. During this period, Hoagland nutrient solution is watered every two weeks to supplement nutrients.

Claims

1. A method for inducing callus and regenerating adventitious buds from Acer truncatum stem segments, characterized in that: The specific steps include: 1) Inoculate the stem segments of Acer truncatum into callus induction medium for 20 days in dark culture at 24±1℃ to obtain Acer truncatum callus; 2) Inoculate the Acer truncatum callus into the adventitious bud differentiation medium for differentiation culture, with a subculture period of 20 days, a culture photoperiod of 12 hours light / 12 hours dark, and a culture temperature of 24±1°C to obtain the Acer truncatum callus that differentiates into adventitious buds; 3) When the adventitious buds generated by callus differentiation grow to 1-2 cm, cut off the adventitious buds and inoculate them into rooting medium for rooting culture. The culture light cycle is 12 hours light / 12 hours dark, and the culture temperature is 24±1℃. After about 1-2 weeks of culture, adventitious roots grow from the base of the adventitious buds, and the Acer truncatum regeneration plants are obtained; The callus induction medium formula is NN69 medium + 30g / L sucrose + 6.5g / L agar + 0.2mg / L 2,4-D + 0.5mg / L BA; or NN69 medium + 30g / L sucrose + 6.5g / L agar + 0.2mg / L IBA + 0.5mg / L BA; The adventitious bud differentiation medium formula is NN69 medium + 30g / L sucrose + 6.5g / L agar + 0.25mg / L CPPU; or NN69 medium + 30g / L sucrose + 6.5g / L agar + 0.5mg / L CPPU; or NN69 medium + 30g / L sucrose + 6.5g / L agar + 0.25mg / L CPPU + 0.25mg / L BA; The rooting medium formula is WPM medium+IBA 0.5 mg / L+30 g / L sucrose+AC 0.5 g / L+6.5 g / L agar.

Images