Method for inducing loose embryogenic callus of arundo donax

Abstract

The application discloses a method for inducing loose-type embryogenic callus of bamboo, and relates to the field of plant tissue culture, and comprises the following steps: (1) using the root of a bamboo tissue culture seedling as an explant, and inducing callus on a 1 / 2MS+2-3mg / L 2,4-D+15g / L sucrose culture medium; (2) cutting the callus from the bamboo explant and transferring the callus to a MS+1-2mg / L 6-BA+1-2mg / L 2,4-D+30g / L sucrose culture medium for induction; (3) transferring the induced loose-type callus of bamboo to a MS+0.2-0.5mg / L 6-BA culture medium, placing the inoculated culture bottle in a constant-temperature incubator with a temperature of 10-15 DEG C for dark culture for 3 days, then transferring the culture bottle to a constant-temperature incubator with a temperature of 20 DEG C for continuous culture for 7 days, then transferring the culture bottle to a MS+0.2-0.5mg / L 6-BA+0.5mg / L 2,4-D+30g / L sucrose culture medium for subculture for 3-5 times; and (4) transferring the culture to a MS+0.1-0.2mg / L 6-BA+0.3-0.5mg / L 2,4-D+30g / L sucrose culture medium for culture. The loose-type embryogenic callus rate is almost 100%, and the loose-type embryogenic callus of bamboo can be kept for more than 6 months.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant tissue culture, in particular to a method for inducing loose type of embryogenic callus of Arundo donax. BACKGROUND

[0002] Arundo donax is a perennial large herbaceous plant of the genus Arundo in the family Poaceae, with a height of up to 10 m in the productive period, and well-developed rhizomes. Arundo donax has strong adaptability and fast growth, and is often used as papermaking and landscape timber. Since the 20th century, under the background of energy shortage and increasing environmental awareness, European countries have screened 20 perennial herbaceous plants, among which Arundo donax is considered to be the most potential perennial grass species for energy plants. In recent years, according to the biological characteristics of Arundo donax, its characteristics in restoring soil degradation, repairing heavy metal pollution in wetlands, and as a new type of biomass energy have been studied, and its utilization value and development potential have also been increasingly valued by people.

[0003] Arundo donax is asexually propagated, and its seeds are sterile, so it is very difficult to use hybridization breeding. The best method to enrich its germplasm resources is to use mutagenic breeding and genetic transformation breeding. In view of the environmental release problem of transgenic plants, the most feasible method at present is to use somatic cell in vitro mutagenic breeding, and the most ideal material for mutagenesis is embryogenic callus.

[0004] Plant cells can be induced to change into callus cells with characteristics of parenchyma cells through dedifferentiation. Such cells generally have poor stability and can easily change into other types of cells under certain conditions, among which they can change into embryogenic callus cells. Such embryogenic callus cells can easily differentiate into embryoid through embryogenesis, and regenerate into complete plants under appropriate conditions. The above regeneration mode of callus through dedifferentiation, redifferentiation, and embryogenesis is often needed in plant cell engineering. Embryogenic callus cells are mainly used in plant material preservation, propagation, genetic transformation, somatic cell in vitro mutagenic breeding, and other directions, and can also be used in basic research of plant molecular genetic mechanisms and cell biology. Therefore, the problem of inducing plant embryogenic callus will often be encountered in production and research.

[0005] During callus induction, the induction conditions can cause different changes in the texture and structure of callus, some of which are tight and some of which are loose, which mainly depends on the characteristics of the linkage between cells that make up the callus mass. When callus cells are tightly linked to each other, the callus mass will appear tight, and conversely, it will show loose structure.

[0006] In general, after the plant callus is induced in vitro, it is further induced to be embryogenic, and finally the callus embryogenesis is achieved. However, when the loose callus of Arundo donax is induced to be loose embryogenic callus, the callus often appears brown and dead, and the probability of embryogenesis is extremely low or even no embryogenesis. Therefore, it is of great significance to establish an efficient embryogenic callus induction system for the germplasm innovation and new variety breeding of Arundo donax. SUMMARY

[0007] The purpose of the present application is to establish a loose embryogenic callus induction system of Arundo donax, and to provide ideal test materials for mutation breeding, tissue detoxification and tissue culture and rapid propagation of Arundo donax.

[0008] The technical scheme adopted to achieve the purpose of the present application is:

[0009] A method for inducing loose embryogenic callus of Arundo donax, mainly comprising the following steps:

[0010] (1) Preliminary induction of Arundo donax callus

[0011] The explant of Arundo donax tissue culture seedling is used to induce callus on the medium, and the culture is carried out at room temperature in the dark, and no subculture is needed during the culture.

[0012] The bamboo stem, branch and root of the Arundo donax tissue culture seedling can be used as the explant, and preferably the root of the Arundo donax tissue culture seedling is used as the explant;

[0013] The medium is 1 / 2MS+2-3mg / L 2,4-D+15g / L sucrose medium;

[0014] The culture condition is: the culture temperature is 24-26℃, the culture is carried out in the dark, the culture time is 30d, and no subculture is needed during the culture.

[0015] The induced callus is in a very soft state, and generally does not need to be cut with a scalpel, but can be separated from the explant with tweezers;

[0016] (2) Induction of loose callus of Arundo donax

[0017] The callus induced in step (1) is cut from the Arundo donax explant and transferred to the medium to induce loose callus. The culture condition is: subculture for several times at room temperature under continuous light;

[0018] The medium used in this step is MS+1-2mg / L 6-BA+1-2mg / L 2,4-D+30g / L sucrose medium;

[0019] Further, the state of the callus is observed when subculturing. The state of the loose type of callus of Arundo is characterized in that the callus mass is composed of granular small calli which are easily dispersed, and the callus mass is hard when subcultured, which is different from the soft type of callus induced in the preliminary induction. The callus mass which becomes brown is discarded, and the callus mass which is hard and white is selected for subculture. The culture is stopped when the callus reaches the above-described state.

[0020] Further, the culture conditions are as follows: the culture temperature is 24-26°C, continuous light culture, and the light intensity is 500-1000 Lux. The step needs 2-3 times of subculture.

[0021] (3) Induction of the loose type of embryogenic callus of Arundo

[0022] The loose type of callus of Arundo induced in step (2) is transferred to a culture medium to which sucrose is added, and the culture bottle after inoculation is placed in a thermostat incubator at a temperature of 7-20°C for culture in the dark. Preferably, the temperature is 10-15°C.

[0023] Further, the culture medium in the above process is MS+0.2-0.5 mg / L 6-BA, 40 g of sucrose is added to the culture medium, and the culture time is 3 days.

[0024] Then, the culture bottle is transferred to a thermostat incubator at a temperature of 15-25°C for further culture. Preferably, the culture time is 7 days, and the culture temperature is 20°C.

[0025] After the culture, the callus in the culture bottle is subcultured to a new culture medium with sucrose for further culture.

[0026] Further, the new culture medium in the above process is MS+0.2-0.5 mg / L 6-BA+0.35-0.5 mg / L 2,4-D+30 g / L sucrose, and preferably, the culture medium is MS+0.5 mg / L 6-BA+0.5 mg / L 2,4-D+30 g / L sucrose. The culture conditions are as follows: the culture temperature is 24-26°C, continuous light culture, and the light intensity is 500-1000 Lux. The subculture is performed 3-5 times. After 3-5 times of subculture, the ideal loose type of embryogenic callus of Arundo is obtained.

[0027] The identification method of the loose embryogenic callus is as follows: the callus group to be detected is placed on a slide, 1 drop of acetic acid carmine staining agent is dropped, 2-3 minutes later, a cover glass is covered to press the slide, so that the callus cells are dispersed, and observation is conducted under a microscope; when more than 70-80% of the cell nuclei in the observed bamboo callus cells reach a ratio of 0.3-0.4 with the cell diameter, and the proportion of the cells in the observed cells in the mitotic phase reaches more than 1%, it is determined that the induced callus reaches complete embryogenesis, and the morphology of the callus is still in the loose type;

[0028] (4) Bamboo loose embryogenic callus maintenance culture

[0029] The bamboo loose embryogenic callus induced in step (3) is transferred to sucrose culture medium for culture;

[0030] Further, the sucrose culture medium in the step is a culture medium of MS+0.1-0.2 mg / L 6-BA+0.3-0.5 mg / L 2,4-D+30 g / L sucrose;

[0031] The culture condition is as follows: the culture temperature is 22-23℃, continuous light culture is conducted, the light intensity is 500-1000 Lux, the subculture time is 20 d, and after multiple subculture, the embryogenic callus can be maintained for more than 180 d;

[0032] The embryogenic callus is observed under a microscope, and the callus with an appearance not conforming to the loose type and the callus with discoloration is discarded.

[0033] Compared with the prior art, the beneficial effects of the present application are as follows:

[0034] The loose type embryogenic callus of Arundo donax cannot be induced by changing the culture medium only, and cannot be induced by adjusting the temperature only, and the temperature is not matched with the culture medium. After the loose type callus of Arundo donax is induced, the callus is transferred to the specific culture medium MS+0.2-0.5 mg / L 6-BA for culture, and after the culture is finished, the loose type embryogenic callus is induced by the method of low temperature stress combined with hormone induction, that is, 10-15 DEG C for callus culture, and the following technical features are combined: the callus culture bottle is transferred to a constant temperature incubator with a temperature of 20 DEG C for continuous culture, after the culture is finished, the callus in the culture bottle is transferred to a new culture medium MS+0.2-0.5 mg / L 6-BA+0.5 mg / L 2, 4-D+30 g / L sucrose for continuous culture, the callus microscopic examination standard is that when the percentage of cells in the division stage in the total number of cells in the total observation field of view is more than 1%, the next culture is carried out, the induction conditions are adjusted and the embryogenic state of the callus is judged by combining the microscope examination technology, and finally the induction rate is almost 100%, the induction is high, and the induction result is complete embryogenesis. By the method, the embryogenic change of the callus is greatly affected by low temperature and short time treatment, and the induction time and the induction rate are promoted.

[0035] The selection of the explant is that because the meristem of the root of Arundo donax is relatively more, the embryogenic callus is induced, and the explant is easy to prepare and simple to operate.

[0036] The induction of the loose type embryogenic callus of Arundo donax is different from other plants, and the loose type embryogenic callus cannot be successfully cultured by the conventional culture mode, increasing the addition amount of each hormone in the culture medium or adjusting the proportion or type of the hormone, and the normal temperature, and the loose type embryogenic callus can be successfully induced only by the specific temperature, the specific culture medium and the specific addition amount of the hormone, and the three are indispensable, and the growth speed is fast.

[0037] The structure of the callus mass directly determines the characteristics of the callus during embryogenesis and embryoid formation. Generally, the embryogenic efficiency of callus with loose structure is greatly improved because the cells are not closely connected, which does not cause differences between the inner and outer environments of the callus mass, and finally makes each cell in the callus mass receive the same influence during embryogenesis, so that each cell has a similar degree of embryogenesis, and most cells in the callus mass can achieve embryogenesis according to the processing target. After the loose callus cells are embryogenetic, the connection between the cells remains unchanged, and finally the embryogenesis is mainly single cell embryogenesis. Single cell embryogenesis can greatly improve the efficiency of various cell engineering applications and can bring revolutionary changes, which has a wide and far-reaching effect. The callus induced in the present application has a loose structure, and the callus mass can be easily pressed into a single layer of cells by tabletting, and the cells are spherical and thin-walled.

[0038] The main difficulty in the induction process of loose embryogenic callus is the embryogenesis of callus cells, that is, how to convert a dedifferentiated plant cell into a meristematic parenchyma cell with embryonic characteristics. The present patent mainly uses a low temperature stress method combined with certain tissue culture conditions, and establishes a high-efficiency loose embryogenic callus induction system of Arundo donax, which lays a foundation for Arundo donax somatic mutation breeding, tissue detoxification and tissue culture and rapid propagation.

[0039] The loose embryogenic callus of Arundo donax obtained by the method can be maintained for more than 6 months and can be used for various researches and applications. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Figure 1 is a graph of the induction results of loose embryogenic callus of Arundo donax in the treatment 1 of the comparative example 2 of the present application;

[0041] Figure 2 Figure 2 is a graph of the induction results of loose embryogenic callus of Arundo donax in the treatment 2 of the comparative example 2 of the present application;

[0042] Figure 3 Figure 3 is a graph of the induction results of loose embryogenic callus of Arundo donax in the treatment 3 of the comparative example 2 of the present application;

[0043] Figure 4 Figure 4 is a graph of the induction results of loose embryogenic callus of Arundo donax in the treatment 4 of the comparative example 2 of the present application;

[0044] Figure 5 Figure 5 is a graph of the induction results of loose embryogenic callus of Arundo donax in the treatment 5 of the comparative example 2 of the present application;

[0045] Figure 6 Figure 6 is a graph of the induction results of loose embryogenic callus of Arundo donax in the treatment 6 of the comparative example 2 of the present application;

[0046] Figure 7 Figure of induction result of treatment 7 of Example 2 of the present application on loose-type embryogenic callus of Arundo donax;

[0047] Figure 8 Figure of induction result of treatment 8 of Example 2 of the present application on loose-type embryogenic callus of Arundo donax;

[0048] Figure 9 Figure of induction result of treatment 9 of Example 2 of the present application on loose-type embryogenic callus of Arundo donax;

[0049] Figure 10 Figure of induction result of treatment 10 of Example 2 of the present application on loose-type embryogenic callus of Arundo donax;

[0050] Figure 11 Figure of induction result of treatment 11 of Example 2 of the present application on loose-type embryogenic callus of Arundo donax;

[0051] Figure 12 Figure of induction result of treatment 12 of Example 2 of the present application on loose-type embryogenic callus of Arundo donax;

[0052] Figure 13 Figure of induction result of treatment 13 of Example 2 of the present application on loose-type embryogenic callus of Arundo donax;

[0053] Figure 14 Figure of induction result of loose-type embryogenic callus of Arundo donax at 5-7℃ in Example 3 of the present application;

[0054] Figure 15 Figure of induction result of loose-type embryogenic callus of Arundo donax at 7-9℃ in Example 3 of the present application;

[0055] Figure 16 Figure of induction result of loose-type embryogenic callus of Arundo donax at 10-15℃ in Example 3 of the present application;

[0056] Figure 17 Figure of induction result of loose-type embryogenic callus of Arundo donax at 16-20℃ in Example 3 of the present application;

[0057] Figure 18 Figure of regenerated shoots induced from loose-type embryogenic callus of Arundo donax; DETAILED DESCRIPTION

[0058] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0059] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other in the case of no conflict.

[0060] Embodiment 1

[0061] As shown in the figure, a method for inducing loose-type embryogenic callus of reed mainly comprises the following steps: Figure 1

[0062] (1) Preliminary induction of reed callus

[0063] The root of the reed tissue culture seedling is used as an explant, and callus is induced on a culture medium of 1 / 2MS+2-3mg / L 2,4-D+15g / L sucrose. The culture condition is: culture temperature is 24-26℃, culture in the dark, and culture time is 30d, and no subculture is needed during the period. The induced callus should be in a very soft state, and generally no dissecting knife is needed for cutting, and the callus can be separated from the explant directly by using forceps to clamp it;

[0064] (2) Induction of loose-type callus of reed

[0065] The callus induced in the above step is cut from the reed explant, and is transferred to a culture medium of MS+1-2mg / L 6-BA+1-2mg / L 2,4-D+30g / L sucrose to induce loose-type callus. The culture condition is: culture temperature is 24-26℃, continuous light culture, and light intensity is 500-1000Lux. This step needs subculture for 2-3 times, and attention should be paid to the state of the callus during subculture. The state characteristics of the loose-type callus of reed are: the callus mass is composed of granular small callus, which is easy to disperse, and at the same time, the texture is relatively hard when being transferred, which is different from the soft callus induced in the preliminary induction. The callus mass which becomes brown is discarded during each subculture, and the callus mass which is hard in texture and white in color is selected for subculture. When the callus reaches the state described above, the culture is stopped;

[0066] (3) Induction of loose-type embryogenic callus of reed

[0067] ​The induced loose-type callus of reed canary grass is transferred to MS + 0.2-0.5 mg / L 6-BA medium, 40 g sucrose is added to the medium, the inoculated culture bottle is placed in a constant temperature incubator with a temperature of 10-15°C for culture, no light is needed, the culture time is 3 days, then the callus culture bottle is transferred to a constant temperature incubator with a temperature of 20°C for continuous culture for 7 days, after the culture is finished, the callus in the culture bottle is transferred to new MS + 0.5 mg / L 6-BA + 0.5 mg / L 2,4-D + 30 g / L sucrose medium for continuous culture, the culture condition is: culture temperature 24-26°C, continuous light culture, light intensity 500-1000 Lux. During the period, 3-5 times of subculture can obtain ideal loose-type embryogenic callus of reed canary grass.

[0068] The identification method of loose-type embryogenic callus of reed canary grass: the callus group to be detected is placed on a glass slide, 1 drop of acetic acid carmine staining agent is dropped, 2-3 minutes later, a cover glass is covered for pressing, so that the callus cells are dispersed, observation is carried out under a microscope, when more than 70-80% of the cell nuclei in the observed reed canary grass callus cells reach the ratio of 0.3-0.4 between the cell diameter, and the proportion of cells in mitosis in the observed cells reaches more than 1%, it can be determined that the induced callus reaches complete embryogenesis, and the morphology of the callus is still in loose type; the induction conditions are adjusted and the embryogenic state of the callus is judged by combining the microscope examination technique, finally the induction rate almost reaches 100%, the induction is high, and the growth speed is fast, the structure and texture of the callus are loose structure and hard texture, and the embryogenic state of the callus is all embryogenic.

[0069] (4) Maintenance culture of loose-type embryogenic callus of reed canary grass

[0070] The induced loose-type embryogenic callus of reed canary grass is transferred to MS + 0.1-0.2 mg / L 6-BA + 0.3-0.5 mg / L 2,4-D + 30 g / L sucrose medium for culture, the culture condition is: culture temperature 22-23°C, continuous light culture, light intensity 500-1000 Lux. The subculture time is 20 days, when the appearance of the callus does not conform to the loose-type callus (the standard of being easily pressed into single layer cells by the micro pressing technique is used for identification), and the callus is discarded, and the embryogenic state of the callus is often observed by micro observation.

[0071] Under the above measures, the loose-type embryogenic callus of reed canary grass is maintained for more than 6 months, and is used for various researches and applications.

[0072] Comparative example 1

[0073] Test method: induction of loose-type embryogenic callus of reed canary grass

[0074] The induced loose-type callus of Arundo was transferred to MS+0.5mg / L 6-BA medium, MS+0.5mg / L 2,4D medium, MS+0.5mg / L KT medium for loose-type embryogenic callus induction, 40g sucrose was added to the medium, induction was carried out at three temperatures, the three temperatures were 7℃-10℃, 10℃-15℃, 20℃-23℃ respectively, no light was needed, the culture time was 3d, then the callus culture bottle was transferred to a constant temperature incubator with a temperature of 20℃ for continuous culture for 7d, after the culture ended, the callus in the culture bottle was transferred to new MS+0.5mg / L 6-BA+0.5mg / L 2,4-D+30g / L sucrose medium for continuous culture, the culture condition was: culture temperature 24-26℃, continuous light culture, light intensity 500-1000Lux. The results after 3-5 times of subculture were as follows:

[0075] Table 1 Induction results of different media combined with different induction temperatures

[0076]

[0077] Among them, "+" means that the callus is completely embryogenic; "+-" means that the callus is partially embryogenic; "-" means that the callus is non-embryogenic.

[0078] From the above results, it can be seen that the induction results of loose-type embryogenic callus of Arundo do not become better with the decrease or increase of temperature, and not all hormones are suitable for low-temperature induction method, using 6-BA combined with low-temperature induction (10℃-15℃) combined with later transfer culture, completely embryogenic loose-type callus can be finally cultivated, KT and 2,4-D combined with low-temperature induction method cannot induce loose-type embryogenic callus of Arundo.

[0079] Comparative Example 2

[0080] The loose-type callus induced in step (2) of Example 1 was transferred to a MS + 0.2-0.5 mg / L 6-BA medium with 40 g of sucrose added thereto, and the inoculated culture bottle was placed in a thermostat incubator at a temperature of 10-15°C for culture without light for 3 days, and then the callus culture bottle was transferred to a thermostat incubator at a temperature of 20°C for further culture for 7 days. After the culture, the callus in the culture bottle was transferred to a new 1 / 2MS medium containing different 6-BA, 2,4D and KT for loose-type embryogenic callus induction. Five bottles were inoculated for each test treatment, and the test was repeated three times. Subculture was performed every 15 days, and the medium was not changed. At the time of subculture, loose-type callus was selected for subculture transfer. After three subcultures, the callus induction for each test treatment was counted.

[0081] Table 2 Influence of different hormone ratios on induction of loose-type embryogenic callus of Arundo donax

[0082]

[0083]

[0084] In the table, "+" means that the callus is entirely embryogenic; "+-" means that the callus is partially embryogenic; and "-" means that the callus is non-embryogenic.

[0085] From the above results, it can be seen that after the callus culture bottle was transferred to a thermostat incubator for further culture, the callus in the culture bottle was transferred to a new 1 / 2MS medium containing different 6-BA, 2,4D and KT for loose-type embryogenic callus induction. In the medium containing 6-BA and 2,4-D, entirely embryogenic callus was obtained. In the medium containing only one of 6-BA or 2,4-D, embryogenic callus was not obtained. Some hormones such as KT limited the embryogenic callus induction to be entirely non-embryogenic. The growth rate of most callus was faster with more added hormones, but was not directly related to the embryogenic state of the callus. Only when 6-BA was 0.2-0.5 and 2,4-D was 0.5, did the loose-type embryogenic callus of Arundo donax become embryogenic. When 6-BA and 2,4-D were both 0.5, entirely embryogenic callus was obtained. With a higher concentration of 2,4-D, the growth rate was greatly reduced, and the culture result was non-embryogenic.

[0086] Comparative Example 3

[0087] The induction of the loose type of embryogenic callus of Arundo donax was carried out in the manner of Comparative Example 2, and the induction temperature in step 3 was changed, i.e. the loose type of callus induced in step (2) was transferred to a culture medium, sucrose was added to the culture medium, and the inoculated culture bottles were placed in constant temperature incubators with temperatures of 5-7℃, 7-9℃, 10-15℃, and 16-20℃, respectively, for culture without light. The results are as follows:

[0088] Table 3 Influence of different temperatures on the induction of loose type of embryogenic callus of Arundo donax

[0089]

[0090]

[0091] As shown in the above table, the optimal culture temperature is 10-15℃.

[0092] Comparative Example 4

[0093] The amount of sucrose added was changed on the basis of Comparative Example 1, and the original amount of sucrose added was 40g. By adding 30g and 50g, respectively, the results showed that the induction rate of embryogenic callus did not become better with the increase of the amount of sucrose, and the optimal amount of sucrose added in the third step for the induction of loose type of callus of Arundo donax was 40g.

[0094] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A method for inducing loose-type embryogenic callus of Arundo donax, characterized by, The method comprises the following steps: (1) preliminary induction of Arundo donax callus Bamboo stems or branches or roots of the Arundo donax tissue culture seedlings are used as explants; The explants of the Arundo donax tissue culture seedlings are used to induce callus on a culture medium, and the culture is carried out at room temperature in the dark; the culture medium is a culture medium of 1 / 2MS + 2-3 mg / L 2,4-D + 15 g / L sucrose; The culture conditions are as follows: the culture temperature is 24-26℃, the culture is carried out in the dark, and the culture time is 30 d; (2) induction of loose callus of Arundo donax The callus induced in step (1) is cut from the Arundo donax explants and transferred to a culture medium to induce loose callus; The culture medium used in this step is a culture medium of MS + 1-2 mg / L 6-BA + 1-2 mg / L 2,4-D + 30 g / L sucrose; Hard and white callus groups are selected for subculture each time; The culture conditions are as follows: the culture temperature is 24-26℃, the culture is carried out under continuous light, and the light intensity is 500-1000 Lux; this step needs to be subcultured for several times; (3) induction of loose embryogenic callus of Arundo donax The loose callus of Arundo donax induced in step (2) is transferred to a culture medium of MS + 0.2-0.5 mg / L 6-BA, and 40 g of sucrose is added to the culture medium, and the culture time is 3 d; The inoculated culture bottle is placed in a constant-temperature incubator with a temperature of 7-20℃ and cultured in the dark; Then the culture bottle is transferred to a constant-temperature incubator with a temperature of 15-25℃ and cultured for another 7 d; After the culture is completed, the callus in the culture bottle is transferred to a culture medium of MS + 0.2-0.5 mg / L 6-BA + 0.35-0.5 mg / L 2,4-D + 30 g / L sucrose and cultured continuously, and the culture conditions are as follows: the culture temperature is 24-26℃, the culture is carried out under continuous light, the light intensity is 500-1000 Lux, the subculture is carried out for 3-5 times, and the subculture time is 20 d.

2. The method of claim 1, wherein the induction of the loose-type embryogenic callus of Arundo donax is characterized by: In step (3), the inoculated culture bottle is placed in a constant-temperature incubator with a temperature of 10-15℃ and cultured in the dark; Then the culture bottle is transferred to a constant-temperature incubator with a temperature of 20℃ and cultured; After the culture is completed, the callus in the culture bottle is transferred to a culture medium of MS + 0.5 mg / L 6-BA + 0.5 mg / L 2,4-D + 30 g / L sucrose and cultured continuously.

3. The method according to claim 1 or 2, characterized in that: After the induction of loose embryogenic callus of Arundo donax in step (3), step (4) of maintaining culture of loose embryogenic callus of Arundo donax is carried out: The loose embryogenic callus of Arundo donax induced in step (3) is transferred to a culture medium of MS + 0.1-0.2 mg / L 6-BA + 0.3-0.5 mg / L 2,4-D + 30 g / L sucrose and cultured, and the culture conditions are as follows: the culture temperature is 22-23℃, the culture is carried out under continuous light, and the light intensity is 500-1000 Lux.

4. The method of claim 1 or 2, wherein the method is characterized by: The roots of the Arundo donax tissue culture seedlings are used as explants.

5. The method for inducing loose embryogenic callus of *Arundo donax* according to claim 1 or 2, characterized in that: The subculture is carried out for 2-3 times.

6. A method for identifying loose embryogenic callus of *Arundinaria lobata*, applied to the induction process of loose embryogenic callus of *Arundinaria lobata* as described in claim 1 or 2, characterized in that: The identification method after the step (3) loose type of reed embryogenic callus induction includes the following steps: taking the callus group to be detected on the glass slide, dropping 1 drop of acetic acid magenta dye, covering the cover glass after 2-3 minutes to make the callus cells dispersed, and observing under the microscope. When the observed reed callus cell nucleus and cell diameter ratio reaches 0.3-0.4, and the observed cell in mitosis accounts for more than 1%, the induced callus reaches complete embryogenesis, and the morphology of the callus is still loose.

Images