High-efficiency tissue culture and rapid propagation method of arundo donax

Abstract

The application discloses a high-efficiency reed tissue culture and rapid propagation method, and mainly relates to the reed propagation technical field. The method comprises the following steps: S1, proliferating and culturing sterile explants to obtain reed tissue culture seedlings, and repeatedly subculturing the reed tissue culture seedlings to obtain reed bud clusters; S2, after the reed bud clusters are cut, the reed bud clusters are inoculated on seedling culture medium, the culture temperature is 25-28 DEG C, the light intensity is 2000-3000 Lux, and continuous light culture is carried out for 30-40 days, so that the reed tissue culture seedlings with roots are obtained, and the seedling culture medium is MS culture medium added with 0.05-0.15 mg / L NAA, 1-3 g / L NH4NO3, 0.2-0.8 g / L activated carbon and 10-30 g / L sucrose; and S3, transplanting the reed tissue culture seedlings obtained in S2 to obtain reed plants. The application has the beneficial effects that the nutrition and culture conditions of the seedling stage are strengthened, the growth cycle of the reed seedlings is shortened, and the quality of the seedlings is greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the bamboo reproduction technical field, specifically to a high-efficiency bamboo tissue culture and rapid propagation method. BACKGROUND

[0002] Tissue culture and rapid propagation is an effective means and approach for rapid propagation of seedlings, which has been widely used in seedling production of various plants, and is not only efficient, but also greatly improves the quality of seedlings. In actual production, although tissue culture seedlings have obvious advantages, compared with seedlings, the main disadvantage is that the growth potential is weak during the seedling stage, and a certain period of hardening and a longer period of seedling culture are required, thereby prolonging the culture period and causing the production cost to rise. Therefore, how to shorten the seedling culture time, improve the survival rate and growth quality of the transplanting link, is the difficulty and pain point of tissue culture and rapid propagation technology.

[0003] Bamboo belongs to monocotyledonous plants, and also has the above-mentioned tissue culture problems. At the same time, due to its fast growth characteristics, during the tissue culture seedling stage, the seedlings produced by tissue culture and rapid propagation grow too fast, which leads to the problem of overgrowth of tissue culture seedlings, specifically, the growth potential of tissue culture seedlings becomes weak, the leaves turn yellow, and the growth momentum is insufficient, thereby further exacerbating the long production cycle of tissue culture seedlings during the seedling stage and increasing the production cost. SUMMARY

[0004] The purpose of the present application is to provide a high-efficiency bamboo tissue culture and rapid propagation method, which strengthens the nutrition and culture conditions during the seedling stage, shortens the growth period of bamboo seedlings, and greatly improves the quality of seedlings.

[0005] In order to achieve the above-mentioned purpose, the present application is realized by the following technical scheme:

[0006] A high-efficiency bamboo tissue culture and rapid propagation method, comprising the following steps:

[0007] S1, proliferating and culturing sterile explants to obtain bamboo tissue culture seedlings, and obtaining bamboo bud clusters based on repeated subculture of the tissue culture seedlings;

[0008] S2, after the bamboo bud clusters are cut, they are inoculated on a seedling culture medium, and the culture temperature is 25-28 DEG C, the light intensity is 2000-3000 Lux, and the continuous light culture is carried out for 30-40 days, so that the bamboo tissue culture seedlings with roots are obtained, and the seedling culture medium is MS medium added with 0.05-0.15 mg / L NAA, 1-3 g / L NH4NO3, 0.2-0.8 g / L activated carbon and 10-30 g / L sucrose;

[0009] S3, transplanting the bamboo tissue culture seedlings obtained in S2 to obtain bamboo plants.

[0010] Preferably, the S1 carries out the proliferation culture for the explant, which is two-step proliferation, including the following steps:

[0011] The aseptic explant is carried out the first proliferation culture for 30-40 days, and the MS medium with 0.1-0.2 mg / L 6-BA, 0.2-0.3 mg / L IAA and 30 g / L sucrose is used, and the culture condition is 25-27℃ and 1500-2000 Lux continuous light.

[0012] The first proliferation culture of the bamboo sprout is cut into single bamboo sprout, and is transferred to the MS medium with 0.1-0.2 mg / L IAA and 30 g / L sucrose for the second proliferation culture for 20-30 days, and the culture condition is 25-27℃ and 1500-2000 Lux continuous light; and the bamboo tissue culture seedlings with the proliferation coefficient of 6-9 can be obtained.

[0013] Further, in the two-step proliferation, the first proliferation uses the MS medium with 0.2 mg / L 6-BA, 0.3 mg / L IAA and 30 g / L sucrose, and the second proliferation uses the MS medium with 0.1 mg / L IAA and 30 g / L sucrose.

[0014] Further, the bamboo tissue culture seedlings obtained by the second proliferation culture are selected for the growth of healthy and dark green leaves, and are placed in the MS medium with 10 g / L sucrose for the preservation culture, and after 40-60 days, the dominant plants are selected for the subculture, and the bamboo original seedlings are preserved.

[0015] Further, in the step S1, when the proliferation coefficient of the bamboo sprout is decreased to below 5, the explant is discarded, and the preserved bamboo original seedlings are selected for the new round of proliferation culture.

[0016] Preferably, the culture medium in the S2 is the MS medium with 0.1 mg / L NAA, 2 g / L NH4NO3, 0.5 g / L activated carbon and 20 g / L sucrose, and the thickness is twice of the proliferation culture medium in the step S1.

[0017] Preferably, the transplanting operation in the S3 includes: selecting the bamboo tissue culture seedlings obtained in the S2, placing them in the greenhouse environment for 2-3 days for seedling raising, and then transplanting them into the substrate containing grass carbon soil, vermiculite and earthworm soil with the weight ratio of 1:1:1, and culturing them under the conditions of 20-25℃, direct sunlight and 90-95% relative humidity for 30-40 days, so that the obtained bamboo plants have the height of 40-60 cm and can be transplanted to the open field for planting.

[0018] Compared with the prior art, the application has the beneficial effects that:

[0019] Through the innovation of the tissue culture and rapid propagation technology of the reed, the culture period of the reed tissue culture seedlings is shortened from 120-150 days to 60-90 days, and the quality level of the reed tissue culture seedlings is obviously improved by more than one level, so that the growth potential of the reed tissue culture seedlings is improved, the growth period of the reed seedlings is shortened, and the quality of the seedlings is greatly improved. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a reed tissue culture seedling cultured by a conventional method.

[0021] Figure 2 is a reed tissue culture seedling obtained by the technology of the application.

[0022] Figure 3 is a flowchart of an embodiment of the application. DETAILED DESCRIPTION

[0023] The application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the application and not used to limit the scope of the application. In addition, it should be understood that those skilled in the art can make various modifications or changes to the application after reading the content of the application, and these equivalent forms also fall within the scope defined by the application.

[0024] In the following examples, the instruments, reagents, materials, etc. are conventional instruments, reagents, materials, etc. known in the art, which can be obtained through a regular commercial channel, unless otherwise specified. In the following examples, the experimental methods, detection methods, etc. are conventional experimental methods, detection methods, etc. known in the art, unless otherwise specified.

[0025] S1. Proliferation of reed tissue culture seedlings

[0026] S1.1 Preliminary culture of reed tissue culture seedling proliferation

[0027] The lateral buds of the reed growing healthily and free of diseases and pests in the field are selected as explants, and the aseptic explants are obtained through surface sterilization;

[0028] The reed explants with growth points are transferred to the No. 1 culture medium for primary proliferation culture, the culture time is 30-40 days, and the reed bud clusters after primary proliferation culture are obtained.

[0029] The culture conditions for primary proliferation culture are as follows: the culture temperature is 25-27℃, and the light intensity is 1500-2000 Lux continuous light.

[0030] Culture medium No. 1 is: MS + 0.2 mg / L 6-BA + 0.3 mg / L IAA + 30 g / L sucrose.

[0031] S1.2 Second step of proliferation culture of reed tissue culture seedlings

[0032] The reed bud clumps obtained in step s1 are cut into single buds with scissors, and the separated reed buds are transferred to culture medium No. 2 for secondary proliferation culture, and the culture conditions are the same as those of the primary proliferation culture, and the culture is carried out for 20-30 days to obtain the proliferated reed tissue culture seedlings.

[0033] The obtained reed tissue culture seedlings are repeatedly subcultured for many times, and the proliferation coefficient of the reed bud clumps obtained after each subculture is 6-9. The obtained reed bud clumps can be directly subjected to the one-step seedling culture of step S2.

[0034] When the proliferation coefficient is reduced to below 5, the explants are discarded, and the preserved reed original seedlings are reselected for a new round of proliferation culture;

[0035] Culture medium No. 2 is: MS + 0.1 mg / L IAA + 30 g / L sucrose.

[0036] The culture conditions for secondary proliferation culture are: culture temperature 25-27℃, light intensity 1500-2000 Lux continuous light.

[0037] S1.3 Selection and preservation of reed original seedlings

[0038] In step S1.2, among the tissue culture seedlings subjected to secondary proliferation culture, the tissue culture seedling plants with healthy growth and dark green leaves are selected and placed in the preservation medium for preservation culture of reed original seedlings. After 40-60 days of subculture, the dominant plants are selected for subculture. The above method can preserve the reed tissue culture seedling original seedlings for up to 2 years.

[0039] The preservation culture conditions are: culture temperature 15-18℃, light intensity 1500-2000 Lux continuous light.

[0040] The preservation medium is: MS + 10 g / L sucrose.

[0041] S2. One-step seedling culture of reed tissue culture seedlings

[0042] The reed bud clumps obtained in S1.2 are cut into single buds with scissors in a clean bench, and are inoculated into the seedling culture medium for culture. After 30-40 days of culture, healthy and rooted reed tissue culture seedlings are obtained, which can be directly used for transplanting of test tube seedlings.

[0043] The seedling culture medium is: MS medium + 0.1 mg / L NAA + 1-3 g / L NH4NO3+ 0.5 g / L activated carbon + 20 g / L sucrose, and the thickness of the seedling culture medium is 1 times the thickness of the proliferation culture medium (No. 1 medium, No. 2 medium).

[0044] The seedling culture conditions are: culture temperature 25-28℃, light intensity 2000-3000 Lux continuous light.

[0045] S3. Transplanting of reed tissue culture seedlings

[0046] Among the reed tissue culture seedlings obtained in S2, the reed tissue culture seedlings to be transplanted are opened and placed in a transplanting greenhouse environment for hardening for 2-3 days, and then transplanted into a substrate containing grass charcoal + vermiculite + earthworm soil (weight ratio 1:1:1) for culture. The culture conditions are: temperature 20-25℃, direct sunlight irradiation, and relative humidity maintained at 90-95%. After 30-40 days, the plants can grow to 40-60 cm high, and can be transplanted to the open field for planting.

[0047] Through the above method, the culture period of reed tissue culture seedlings is shortened from 120-150 days to 60-90 days, and the quality level of tissue culture seedlings is obviously improved by more than 1 level. For specific effects, please refer to the attached Figure 1 、 2 , wherein Figure 1 is a reed tissue culture seedling cultured by a conventional method, Figure 2 is a reed tissue culture seedling obtained by using the improved method.

[0048] After the improvement, the rapid propagation technology has made breakthroughs in the following aspects:

[0049] First, the use of cytokinin. Cytokinin is generally used for proliferation culture of tissue culture seedlings, but long-term and high-concentration use will cause the growth of plants to be affected by it for a long time. In reed tissue culture, the main performance is that during the seedling stage of reed, the roots of the tissue culture seedlings will still produce a large number of proliferation buds, although it will not have a great impact on the tissue culture seedlings, but it will cause the growth potential to decrease and the growth rate to slow down. In addition, during the transplanting and in-ground process of the plants, sprouting occurs from time to time, which also slows down the growth rate of the seedlings and reduces the production efficiency of the tissue culture seedlings.

[0050] The study adopts one-time addition of cytokinin, maintains the proliferation of tissue culture seedlings by using the residual hormones in the plant, and uses the preservation of original seedlings to timely supplement the proliferation rate of tissue culture seedlings, so as to continuously maintain a certain proliferation efficiency. The proliferation culture and tissue culture seedling culture are separated, and the one-time addition of cytokinin is used. According to the change of the proliferation coefficient, cytokinin is supplemented in time during the proliferation process. This kind of tissue culture process can reduce the time of cytokinin existing in the tissue culture seedlings, slow down the effect of continuous influence of cytokinin on the tissue culture seedlings, ensure the division, and also well control the negative influence of cytokinin. It is an innovation in the tissue culture and rapid propagation; in addition, reducing the proliferation coefficient of tissue culture seedlings can reduce the use of cytokinin, thereby reducing the problem of cytokinin affecting the sprouting of bamboo tissue culture seedlings; and secondly, it simplifies the difficulty of subculture operation, thereby reducing the labor cost.

[0051] Secondly, the control of the proliferation coefficient can increase the efficiency of tissue culture and rapid propagation. In the study, the proliferation efficiency is controlled at 6-9. Under the condition of such proliferation coefficient, the bamboo proliferation bud can be easily divided, and the single bud with healthy growth can be obtained, which reduces the difficulty of manual operation, speeds up the speed, and can obtain higher tissue culture efficiency.

[0052] Embodiment:

[0053] Based on the above technology, for step S2. One-step seedling culture of bamboo tissue culture seedlings, further grouping test is carried out for different NH4NO3 concentrations. The specific test method is as follows:

[0054] Preparation of culture medium:

[0055] The seedling culture medium is MS+NAA 0.1mg / L+NH4NO3+0.5g / L activated carbon+20g / L sucrose+2.37g / L agar (pH 5.8), wherein NH4NO3 is taken as 0, 1, 2, 3mg / L different concentrations, and different seedling culture media are configured, 19 bottles for each culture medium.

[0056] After the second proliferation culture in step S1.2, the bamboo bud clusters obtained are divided into single buds in the clean bench, and are inoculated into the prepared above-mentioned seedling culture medium, and are cultured under the condition of temperature (25±2) ℃, light time 24h, light intensity 2000lx. The material is regularly observed and recorded to record the thickness and growth of the tissue culture seedlings. The experimental results are shown in Table 1.

[0057] Table 1: Test results of one-step seedling culture under different NH4NO3 concentrations

[0058]

[0059] From Table 1, it can be seen that the addition of NH4NO3 in the medium is obviously better than the control without NH4NO3, and the plant height and leaf width are obviously increased. From the dosage of NH4NO3, the treatment of adding 2 g / L is the best for the growth of the tissue culture seedlings of Arundo donax.

Claims

1. An efficient tissue culture micropropagation method of Arundo donax, characterized by, The method comprises the following steps: S1, proliferating culture of the aseptic explants to obtain bamboo cane tissue culture seedlings, and repeatedly subculturing the bamboo cane tissue culture seedlings to obtain bamboo cane buds; S2, after the bamboo cane buds are cut, the bamboo cane buds are inoculated on a seedling culture medium, and the seedling culture medium is composed of 0.05-0.15 mg / L NAA, 1-3 g / L NH4NO3, 0.2-0.8 g / L activated carbon, 10-30 g / L sucrose and MS culture medium; the culture temperature is 25-28 DEG C, the light intensity is 2000-3000 Lux, and continuous light culture is carried out for 30-40 days to obtain bamboo cane tissue culture seedlings with roots; S3, the bamboo cane tissue culture seedlings obtained in S2 are transplanted to obtain bamboo cane plants; The proliferating culture of the aseptic explants in S1 is two-step proliferation, which comprises the following steps: The aseptic explants are proliferated and cultured for 30-40 days to obtain first proliferated and cultured bamboo cane buds; the culture medium used is composed of 0.1-0.2 mg / L 6-BA, 0.2-0.3 mg / L IAA, 30 g / L sucrose and MS culture medium; and the culture conditions are that the temperature is 25-27 DEG C and the light intensity is 1500-2000 Lux. The first proliferated and cultured bamboo cane buds are cut into single bamboo cane buds, and the single bamboo cane buds are transferred to a culture medium composed of 0.1-0.2 mg / L IAA and 30 g / L sucrose and MS culture medium for secondary proliferating culture for 20-30 days; the culture conditions are that the temperature is 25-27 DEG C and the light intensity is 1500-2000 Lux; and bamboo cane tissue culture seedlings with a proliferation coefficient of 6-9 can be obtained.

2. The method according to claim 1, wherein, In the two-step proliferation, the culture medium used in the first proliferation is composed of 0.2 mg / L 6-BA, 0.3 mg / L IAA, 30 g / L sucrose and MS culture medium; and the culture medium used in the second proliferation is composed of 0.1 mg / L IAA, 30 g / L sucrose and MS culture medium.

3. The method of claim 1, wherein the method is characterized by, The bamboo cane tissue culture seedlings obtained by the secondary proliferating culture are selected, and the bamboo cane tissue culture seedlings with strong growth and dark green leaves are placed in a MS culture medium added with 10 g / L sucrose for preservation culture; after 40-60 days, the dominant plants are selected for subculture to preserve the original bamboo cane seedlings.

4. The method according to claim 3, wherein, In step S1, when the proliferation coefficient of the bamboo cane buds is reduced to below 5, the explants are discarded, and the preserved original bamboo cane seedlings are selected for a new round of proliferating culture.

5. The method of claim 1, wherein the method is characterized by, The seedling culture medium in S2 is composed of 0.1 mg / L NAA, 2 g / L NH4NO3, 0.5 g / L activated carbon, 20 g / L sucrose and MS culture medium, and the thickness is twice that of the proliferating culture medium in S1.

6. The method of claim 1, wherein the method is characterized by, The transplanting operation in S3 comprises the following steps: the bamboo cane tissue culture seedlings obtained in S2 are selected and placed in a greenhouse environment for 2-3 days for seedling raising, and then the bamboo cane tissue culture seedlings are transplanted into a substrate containing grass carbon soil, vermiculite and earthworm soil at a weight ratio of 1:1:1; the substrate is cultured for 30-40 days under the conditions of a temperature of 20-25 DEG C, direct sunlight irradiation and a relative humidity of 90-95%, and bamboo cane plants with a height of 40-60 cm are obtained, which can be transplanted to an open field for planting.

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