CN102499086A - Method for breeding locust - Google Patents

Abstract

The invention discloses a breeding method of Robinia pseudoacacia, using zygotic embryos of different embryonic ages as explants, using MS+2,4-D+BA+sucrose+agar as the induction medium for embryogenic callus; using MS as the basic Culture medium + MES + glutamine + hydrolyzed casein + naphthalene acetic acid + 6-benzylaminoadenine + sucrose + agar is the somatic embryo induction medium, through the induction of callus, after the formation of spherical embryos, transfer them to On MS+hydrolyzed casein medium, the mature culture of somatic embryos was carried out, and then transferred to MS basic medium to germinate and form complete plantlets. In the method of the invention, the callus induction rate, the somatic embryo induction rate and the germination rate are high, and a large number of excellent test-tube plantlets of Robinia pseudoacacia can be formed in a short period of time, and large-scale and factory production can be carried out.

Description

A kind of propagation method of black locust

technical field

The invention relates to a method for plant tissue culture, in particular to a method for in vitro culture and plant regeneration of black locust, and belongs to the field of tissue culture of black locust.

Background technique

Black locust (Robinia pseudoacacia L.), also known as acacia, belongs to the leguminosae (Leguminosae) Papilionaceae Robinia L., a deciduous tree, native to the United States, naturally distributed in the Appalachian Mountains in the eastern United States (Appalachian .Mt.) and the Ozank Mountains (Ozank.Mt.), which were introduced into my country in the early 20th century and rapidly expanded and planted after the introduction, are the main timber forests and firewood forests in the middle and lower reaches of the Yellow River, the Huaihe River Basin, the Haihe River Basin, and the lower reaches of the Yangtze River. , water and soil conservation forests, seawall and river embankment protection forests, there are 4 billion trees in only 6 provinces and cities including Hebei, Henan, Shandong and Shanxi. It grows fast and is an important fast-growing timber tree species. Its wood material is hard, with high compressive strength, strong drought tolerance and strong corrosion resistance. Robinia pseudoacacia roots have a large number of nodules, which can increase soil fertility through biological nitrogen fixation, and play an important role in maintaining ecological balance and improving stand quality. Robinia locust leaves contain crude protein, which is a high-quality and cheap livestock feed resource. Robinia pseudoacacia is rich in nutrients. Although the flowering period is short, the flower quantity is considerable. In addition to direct use, it can also produce nectar and extract spices.

China's breeding research on Robinia pseudoacacia began in the 1970s and has achieved many achievements, but it still cannot meet the needs of forestry production. Somatic embryo regeneration is an important platform for breeding and genetic improvement of trees, and it is also a common regeneration system for gene transformation. Somatic embryogenesis technology has the characteristics of large number of reproduction, fast speed, not limited by kinship, once a somatic embryo with a complete structure can generally germinate directly to form a small plant, it is a stable and efficient plant regeneration system. In addition, somatic embryos originate from a single cell, and their developmental program is similar to that of zygotic embryos. They can be used as a model system for embryological research, and are of great significance to the study of theoretical issues such as cell totipotency expression process and cell differentiation mechanism. Laine E and Dumet D successfully preserved the embryogenic callus of Pine caribensis and oil palm at ultra-low temperature and maintained the potential of somatic embryogenesis. After thawing, they can continue to be cultured to form plants, which provides the possibility to save endangered plants. The in vitro culture research of Robinia pseudoacacia began in the late 1950s and reached its climax after the 1980s.

So far, Robinia pseudoacacia has obtained complete plants through cambium culture, stem culture, leaf culture, and unpollinated ovary culture, etc., but these studies have focused on the organogenesis pathway, and the research on the somatic embryogenesis pathway has not few. For example, Hongyan used the cotyledons of mature seeds of Robinia pseudoacacia as explants to study the somatic embryo culture and induction of adventitious buds in Robinia pseudoacacia, but the highest incidence of somatic embryos was only 36.7%.

A large number of studies have shown that the developmental stage of explants is a key factor affecting somatic embryogenesis, and immature zygotic embryos have significant advantages in inducing somatic embryogenesis because of their high totipotency and easy dedifferentiation. Most plants in vitro culture generally use immature zygotic embryo explants, but the somatic embryo induction rate of too young or over mature zygotic embryos is not ideal. Throughout the development of zygotic embryos, there is only a short stage with strong somatic embryogenic potential. At present, there are many studies on the influence of zygotic embryo development stage on the induction of somatic embryos, but as far as Robinia pseudoacacia is concerned, the somatic embryo culture of Robinia pseudoacacia is difficult, which shows that the induction rate of somatic embryos is low, the number of deformed embryos is large, and the quality of somatic embryos is poor. The high level restricts the degree of its genetic improvement, and also hinders the high-efficiency reproduction and rapid promotion of excellent clones. At present, there are relatively few studies on somatic embryo culture of Robinia pseudoacacia at home and abroad, and there are no related reports in China. Only Merkle et al. and Arrillaga et al. abroad have published research results in this regard. Among them, Merkle et al. selected immature zygotic embryos from 3 black locust trees as explants every other week from the first week after flowering, and used MS medium as the basic medium to study the effect of embryo age on explants. The influence of embryo induction, somatic embryogenesis was only observed from an immature zygotic embryo 4 weeks after flowering, and the induction rate of somatic embryos was low; Arrillaga et al. improved the somatic embryos on the basis of Merkle's research Induction rate, using FM medium as the basic medium, using mature zygotic embryos 1-4 weeks after flowering as explants for somatic embryo induction culture, the test results show that the explants 2-3 weeks after flowering The embryo induction rate was the highest, only reaching 12%. The above-mentioned studies all use the immature zygotic embryos 1-4 after flowering as explants to induce culture, and the induction rate is low, the highest induction rate is only 12%, which is difficult to meet the needs of Robinia pseudoacacia propagation and breeding. Therefore, the establishment of Robinia pseudoacacia efficient Somatic embryogenesis system is imperative.

Contents of the invention

The primary purpose of the present invention is to provide a kind of new method for black locust in vitro culture and plant regeneration at the problem that above-mentioned prior art exists, the somatic embryo induction rate of black locust of this method is high, the germination rate of somatic embryo is high, can be in shorter A large number of excellent black locust test-tube seedlings can be formed within a short period of time, which can be used for large-scale and industrial production, and can be used for research on the transformation system of black locust, and can also be used for research on molecular breeding.

To achieve the above object, the present invention provides a method for propagating black locust on the one hand, comprising the steps carried out in the following order:

1) collect black locust pods;

2) taking out the zygotic embryo after carrying out surface sterilization treatment on the Robinia pseudoacacia pod to obtain the sterile zygotic embryo;

3) inoculating the sterile zygotic embryos on the callus induction medium, performing callus induction culture, and obtaining callus;

4) inoculating the callus on the somatic embryo induction medium, performing somatic embryo induction culture, and obtaining the proembryo culture;

5) inoculating the proembryo culture in the somatic embryo maturation medium to carry out maturation culture of somatic embryos to obtain somatic embryos;

6) Inoculating the mature somatic embryos on the germination medium for germination and culturing to obtain somatic embryo seedlings;

7) Inoculating the somatic embryo seedlings on the strong seedling medium for strong seedling cultivation, promoting the growth of somatic embryo seedlings, and obtaining strong seedlings in test tubes;

8) The strong seedlings in test tubes are hardened and transplanted to obtain the product.

The somatic embryos described in the present invention are referred to as somatic embryos for short.

The embryonic age of Robinia pseudoacacia explants had a significant effect on the callus induction rate, and the ideal embryogenic callus induction results could not be obtained from over-tender or over-mature immature embryos. The present invention has found through a large number of experiments that the embryogenic callus induction effect of black locust pods in the time period from the 25th day to the 75th day after flowering is significantly higher than that of pods in other time periods. The improvement; further experiments found that the collection of pods in the time period from the 45th day to the 65th day after the blooming of black locust was further improved as the propagation effect of the explants. The propagation effect of pods in this period of days as explants has been further improved, and the best propagation effect has been achieved by using black locust pods in the 55th day after flowering as explants: collecting black locust pods on the 55th day after flowering The callus rate of the immature zygotic embryos in the pods of the first day was higher than that of the explants in other periods on all induction media, the callus induction rate was 66.72-95.42%, and the induced callus growth rate was fast , light yellow or light yellow green, compact structure, granular, and can still maintain high vigor after multiple subcultures; its somatic embryo induction rate is 76.88-90.73%, somatic embryo germination rate is 80-100%, are significantly higher than the explants in other periods, so the present invention most preferably collects the pods of Robinia pseudoacacia on the 55th day after flowering as the explants.

Wherein, the sterilization process described in step 2) includes the steps carried out in the following order:

A) after the black locust pods collected are washed with sterile water, after soaking the pods with alcohol, rinse them with sterile water;

B) Soak pods with HgCl _solution , rinse with sterile water;

C) taking out the zygotic embryo from the pod after absorbing the water on the surface of the pod.

In particular, the volume percent concentration of the alcohol described in step A) is 75%; the soaking time is 30s; and the sterile water is rinsed 3-5 times.

In particular, the mass percent concentration of the HgCl ₂ solution in step B) is 0.1%; the soaking time is 3-7 minutes, preferably 5 minutes; and the sterile water is washed 5-6 times.

Wherein, the callus induction medium described in step 3) is MS basic medium+2-(N-morpholine)ethanesulfonic acid (MES) 500mg/L+2,4-dichlorophenoxyacetic acid 0.3 -6mg/L+6-benzylaminoadenine 0-3.0mg/L+sucrose 30g/L+agar 6g/L, the pH value is 5.8; preferably: MS basic medium+MES 500mg/L+2,4-dichloro Phenoxyacetic acid 0.3-5.0mg/L + 6-benzylaminoadenine 0-3.0mg/L + sucrose 30g/L + agar 6g/L, pH value is 5.8; further preferred: MS basic medium + MES 500mg/L +2,4-dichlorophenoxyacetic acid 1.0-5.0mg/L+6-benzylaminoadenine 0.3-1.0mg/L+sucrose 30g/L+agar 6g/L, the pH value is 5.8; more preferably: MS Basic medium + MES 500mg/L + 2,4-dichlorophenoxyacetic acid 5.0mg/L + 6-benzylaminoadenine 0.5mg/L + sucrose 30g/L + agar 6g/L, the pH value is 5.8.

In particular, the callus induction culture in step 3) is carried out under the following conditions: under dark conditions, the culture temperature is 26±2°C.

In particular, the relative humidity during callus induction culture is 60-75%.

Wherein, the somatic embryo induction medium described in step 4) is MS basic medium+MES 500mg/L+glutamine 250mg/L+hydrolyzed casein 500mg/L+naphthalene acetic acid 0.1-2.0mg/L+6-benzylamino gland Purine 0-1.0mg/L+ sucrose 30g/L+ agar 6g/L, pH value 5.8; preferably: MS basic medium+MES 500mg/L+glutamine 250mg/L+hydrolyzed casein 500mg/L+naphthaleneacetic acid 0.1-1.0 mg/L+6-benzylaminoadenine 0.1-1.0mg/L+sucrose 30g/L+agar 6g/L, pH 5.8; further preferred: MS basic medium+MES 500mg/L+glutamine 250mg/L+hydrolysis Casein 500mg/L+ naphthaleneacetic acid 0.5mg/L+ 6-benzylaminoadenine 0.5mg/L+ sucrose 30g/L+ agar 6g/L, the pH value is 5.8.

In particular, the somatic embryo induction culture described in step 4) is carried out under the following conditions: under dark conditions, the culture temperature is 26±2°C.

In particular, the relative humidity during somatic embryo induction culture is 60-75%.

Wherein, the somatic embryo maturation medium described in step 5) is MS basic medium + hydrolyzed casein 500 mg/L + sucrose 30 g/L + agar 6 g/L, and the pH value is 5.8.

In particular, the somatic embryo maturation culture described in step 5) is carried out under the following conditions: the culture temperature is 26±2°C, the light intensity is 1500-2000 lux, and the light cycle is 14-16 hours of light/8-10 hours of darkness.

In particular, the relative humidity during somatic embryo maturation culture is 60-75%.

Wherein, the germination medium described in step 6) is MS basic medium+sucrose 30g/L+agar 6g/L, and the pH value is 5.8.

In particular, the germination culture described in step 6) is carried out under the following conditions: the culture temperature is 26±2° C., the light intensity is 1500-2000 lux, and the light cycle is 14-16 hours of light/8-10 hours of darkness.

In particular, the relative humidity during somatic embryo germination culture is 60-75%.

Wherein, the strong seedling medium described in step 7) is MS basic medium+indole butyric acid (IBA) 0.2mg/L+sucrose 30g/L+agar 6g/L, and the pH value is 5.8.

In particular, the cultivation of strong seedlings in step 7) is carried out under the following conditions: the cultivation temperature is 26±2° C., the light intensity is 1500-2000 lux, and the light cycle is 14-16 hours of light/8-10 hours of darkness.

Especially, the relative humidity is 60-75% during the cultivation of somatic embryonic seedlings and strong seedlings.

Wherein, the hardening and transplanting described in step 8) include the steps carried out in the following order: open the bottle cap of the culture bottle, and harden the seedlings in the transplanting room for 1 day; The agar medium was transplanted into the black locust soilless culture medium for transplanting culture, and then transplanted and cultivated for 5-6 weeks before being colonized in the field.

In particular, it also includes placing the test-tube seedlings under shading of 50% natural light and cultivating them for 5-10 days before opening the bottle cap of the culture bottle.

In particular, the soilless culture medium of Robinia pseudoacacia is composed of perlite and vermiculite, wherein the volume ratio of perlite and vermiculite is 1:1.

In particular, the transplanting culture is under natural light conditions, cultivated for 5-6 weeks

Especially, the temperature for transplanting culture is 25±5° C., and the relative humidity is 56-90%.

The propagation method of Robinia pseudoacacia of the present invention has the following advantages:

1. The present invention utilizes immature zygotic embryos of Robinia pseudoacacia to carry out in vitro rapid propagation, and the basic medium used in each culture stage is MS basic medium, which has a high concentration of inorganic salts, especially the content of nitrate, potassium and ammonium High, it can provide the required mineral nutrients for tissue growth and promote the induction, differentiation and germination of somatic embryos.

2. In the propagation method of the present invention, the propagation efficiency of Robinia pseudoacacia is high, adding plant growth regulator in the callus induction medium is beneficial to the induction and proliferation of the callus of explants, and the callus induction rate is high, which can reach 95.42%

3. In the breeding method of the present invention, Robinia pseudoacacia has a high reproductive efficiency. Adding a plant growth regulator in the somatic embryo induction medium is beneficial to the induction, proliferation, maturation, germination and differentiation of somatic embryos from the callus. The somatic embryos used in the present invention The composition of nutrients in the Robinia pseudoacacia medium is reasonable, the dosage and ratio are appropriate, the cultivation results are highly repeatable, the regeneration system is stable, and the induction rate of somatic embryos is high, which can reach 90.73%.

4. The regenerated black locust seedlings cultivated by the method of the present invention grow robustly, have a high reproductive coefficient, and have a high germination rate of mature somatic embryos, reaching 80-100%; the transplanting survival rate of somatic embryo seedlings is high, reaching 100%, which is industrialized large-scale production A simple and fast technical system for Robinia pseudoacacia plants.

Description of drawings

Fig. 1 is the callus that the immature zygotic embryo of Robinia pseudoacacia is induced to culture;

Fig. 2 is the original embryo culture that black locust callus induction culture obtains;

Fig. 3 is the microscopic observation photograph of the thin layer section of proembryo culture;

Fig. 4 is the initial stage culture that black locust proembryo culture carries out mature culture initial stage and obtains;

Fig. 5 is the mature somatic embryo obtained by maturation culture of Robinia pseudoacacia proembryo culture;

Fig. 6 is the somatic embryo seedling that the mature somatic embryo of Robinia pseudoacacia germinates and cultivates;

Fig. 7 is the clustered test-tube strong seedlings with multiple stem and leaf ends obtained by culturing the somatic embryo seedlings of Robinia pseudoacacia;

Fig. 8 is the test-tube strong seedling with dominant backbone obtained in the subculture process of Robinia pseudoacacia somatic embryo seedling strong seedling culture;

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1

1. Test materials

1, the present invention takes the well-growing diploid Robinia pseudoacacia seedling population of Beijing Yanqing Mijiabao Nursery as a resource for drawing materials, and chooses 10 mother trees that grow well, have straight stems, and have a high seed setting rate (the interval between each strain is greater than 50 meters), between June and September 2010, collect the zygotic embryos of different developmental stages of Robinia pseudoacacia in the 4th to 12th week after flowering as explant test materials, collect once every 10 days, and collect 500 pods each time , that is, collect pods 25, 35, 45, 55, 65, and 75 days after flowering, collect 500 pods each time, store the pods collected on the same day in an ice box at low temperature and bring them back to the laboratory, and then put them in a refrigerator at 4°C Refrigerate for later use.

2. Plant growth regulator

The plant growth regulating substance used in the present invention adopts domestic naphthaleneacetic acid (NAA), 6-benzylaminoadenine (6-BA), 2,4-dichlorophenoxyacetic acid (2,4-D) and indole butyric acid (IBA).

3. Preparation of culture medium:

(1) The composition or preparation method of "MS basic medium";

Table 1 MS medium (Murashige and Skoog, 1962)

After the above MS medium mother solution was prepared, it was stored in a refrigerator at 4°C until use. According to the quantity of the configured culture medium, weigh the required agar and sucrose, pour it into sterile water with 3/4 of the volume of the culture medium to be prepared, and add the required mother liquor of macroelements, mother liquor of trace elements and mother liquor of organic components in sequence, each time Add one and stir thoroughly, and finally add water to make up the final volume of the medium, test the pH of the medium with a pH meter, and adjust the pH to 5.8 with 1mol/L NaOH or 1mol/L HCl.

(2) Callus induction medium: MS basic medium supplemented with 2,4-D 0.3-6.0mg/L, 6-BA 0-3.0mg/L, MES 500mg/L, sucrose 30g/L, agar 6g/L L, the pH value was adjusted to 5.8, and the culture medium was sterilized at a constant temperature of 121°C for 15 minutes.

(3) Somatic embryo induction medium: MS basic medium supplemented with NAA 0.1-2.0mg/L, 6-BA 0-1.0mg/L, MES 500mg/L, glutamine 250mg/L, hydrolyzed casein 500mg/L L, 30 g/L sucrose, 6 g/L agar, adjust the pH value to 5.8, and sterilize the medium at a constant temperature at 121° C. for 15 minutes.

(4) Somatic embryo maturation medium: MS basic medium was supplemented with hydrolyzed casein 500mg/L, sucrose 30g/L, agar 6g/L, and the pH value was adjusted to 5.8. The culture medium was sterilized at 121°C for 15 minutes.

(5) Somatic embryo germination medium: MS basic medium was added with 30 g/L sucrose and 6 g/L agar to adjust the pH value to 5.8, and the medium was sterilized at 121° C. for 15 minutes.

(6) Strong seedling medium for somatic embryonic seedlings: add IBA 0.2mg/L, sucrose 30g/L, agar 6g/L to MS basic medium, adjust the pH value to 5.8, and sterilize the medium at 121°C for 15 minutes .

4. Culture conditions

(1) Culture conditions for callus induction culture: dark conditions, culture temperature (26±2)°C, relative humidity during culture is 60-75%.

(2) Culture conditions for somatic embryo induction culture: a closed tissue culture room, under dark conditions, the culture temperature is 26±2° C., and the relative humidity during the culture process is 60-75%.

(3) Culture conditions for somatic embryo maturation culture: culture temperature is (26±2)°C, daylight light source, light intensity is 1500-2000lx, light cycle is 14-16 hours of light/8-10 hours of darkness, during the culture process The relative humidity is 60-75%.

(4) Culture conditions for somatic embryo germination culture: culture temperature is (26±2)°C, daylight light source, light intensity is 1500-2000lx, light cycle is 14-16 hours of light/8-10 hours of darkness, during the culture process The relative humidity is 60-75%.

(5) Culture conditions for strong seedling cultivation of somatic embryonic seedlings: the culture temperature is 26±2°C, the light intensity is 1500-2000lux, the light cycle is 14-16 hours of light/8-10 hours of darkness, and the relative humidity during the culture process 60-75%.

2. Test method

1. Sterilization of explants

Wash the collected pods on the 25th, 35th, 45th, 55th, 65th, and 75th days after flowering respectively, soak the pods with alcohol with a volume percentage concentration of 75% on the ultra-clean workbench for 30 seconds, and then rinse them with sterile water Several times (3-5 times); then with the mass percent concentration of 0.1% _HgCl solution soak pods 3-7min (preferably 5min), rinse several times (5-6 times) with sterile water; then put the pods The immature seeds, i.e. the immature zygotic embryos, were removed from the pods after blotting the surface moisture on sterile and dry filter paper, and the surface sterilized unripe embryos on the 25th, 35th, 45th, 55th, 65th, and 75th days after flowering were obtained. Mature zygotic embryos, spare;

2. Callus induction culture

1) In the ultra-clean workbench, inoculate the whole body of immature zygotic embryos 25, 35, 45, 55, 65, and 75 days after flowering that were sterilized on the surface as explants in the callus induction medium, and in dark conditions The callus induction culture was carried out under the following conditions, subcultured once every 3-4 weeks, that is, the sterilized immature zygotic embryos were cultured for 3-4 weeks, then removed, and placed in another fresh callus induction medium to continue the callus Induction culture of tissue to maintain sufficient nutrients and water in the medium, wherein the culture temperature is (26±2)°C, 2,4-D used in the callus induction medium is 5.0mg/L, 6- The BA is 0.5mg/L, and the relative humidity is 60-75% during the culture process. After 3 weeks of induction culture, the hypocotyl incision of the zygotic embryo begins to expand, forming callus, and the color of the callus is light yellow or light yellow green , the structure is compact and granular (as shown in Figure 1). After 42 days of culture, the callus induction rate was counted. The data analysis results are shown in Table 2.

In the process of callus induction culture, 10 immature zygotic embryos were inoculated for each treatment, and the treatment was repeated 3 times.

Callus induction rate (%) = number of explants that induced callus / total number of inoculated explants × 100%

Table 2 The results of callus induction culture

3. Somatic embryo induction culture

The callus was divided into small pieces of 0.5 cm × 0.5 cm, inoculated on the somatic embryo induction medium, and the induction culture of Robinia pseudoacacia somatic embryos was carried out under dark conditions, wherein the culture temperature was (26 ± 2) ° C, The 6-BA used in the somatic embryo induction medium is 0.5mg/L, and the NAA is 0.5mg/L. During the somatic embryo induction culture, it is subcultured every 3-4 weeks, that is, after the callus block is cultured for 3-4 weeks Take it out, place it in another fresh somatic embryo induction medium to continue the induction culture of somatic embryos, to keep the culture medium containing sufficient nutrients and water, after 6 weeks of induction culture, at the edge of the callus, you can Embryoid body protrusions of different sizes were observed, distributed in concentrated clusters, and after subcultured twice, a yellow-green proembryo culture (as shown in Figure 2) was obtained, and the number of callus pieces induced to obtain somatic embryos was counted. The induction rate of somatic embryos was calculated, and the results are shown in Table 3.

The thin-layer slice test was carried out on the original embryo culture, that is, the original embryo culture was embedded in agarose and sliced with a Leica UC6 automatic vibrating ultrathin microtome (thickness is 100 μm), then stained with rhodamine staining solution, and the Olympus optical Observation under a microscope, the results of microscopic observations are shown in Figure 3. Microscopic examination results show that the proembryo culture has obvious protruding structures, including multiple somatic embryos at the spherical embryo stage that are concentratedly distributed, indicating that there are somatic cells in the induced proembryo culture. Embryo.

During the induction culture of somatic embryos, 5 pieces of callus (0.5×0.5 cm) were inoculated for each treatment, and the treatment was repeated 3 times.

Induction rate of somatic embryos (%) = number of callus pieces induced from primary embryo culture/total number of inoculated callus pieces × 100%

Table 3 Results of somatic embryo induction culture

4. Somatic embryo maturation culture

Transfer the original embryo culture as a whole to the somatic embryo maturation medium without any exogenous hormones, and carry out the maturation culture of somatic embryos. The culture conditions are as follows: the culture temperature is 26±2°C, and the photoperiod is 16h light /8h dark, the light intensity is 1500-2000Lux, the relative humidity is 60-65% during the culture process, and the somatic embryos are subcultured every 3 weeks during the mature culture process. Cultures were continued in fresh somatic embryo maturation medium. After 4 weeks of culture, the somatic embryos on the proembryo culture grew in size and gradually turned green, forming an organism similar to zygotic embryos (as shown in FIG. 4 ). After subculturing twice, it can be observed that the somatic embryo develops to the cotyledon embryo stage (as shown in FIG. 5 ), and a mature somatic embryo is obtained.

5. Germination and culture of somatic embryos

Separate the mature somatic embryos from the callus, transfer them to the germination medium of somatic embryos, place them under the condition of 26±2°C, the photoperiod is 16h light/8h dark, and the light intensity is 1500-2000Lux Culture under conditions, the relative humidity in the germination culture process is 60-75%, the hypocotyl of mature somatic embryos elongates rapidly, and after 4 weeks of culture, the cotyledon at the top of mature somatic embryos unfolds, and the other end differentiates into obvious roots At the growth end, somatic embryo seedlings are formed (as shown in Figure 6), and the somatic embryo seedlings obtained are counted to calculate the germination rate of somatic embryos. The analysis results are shown in Table 3.

During the germination and culture of somatic embryos, 10 somatic embryos were inoculated for each treatment, and the treatment was repeated 3 times.

Germination rate of somatic embryos (%) = number of germinated somatic embryos/number of inoculated somatic embryos × 100%

6. Strong seedling cultivation of somatic embryo seedlings

Transfer the somatic embryonic seedlings to the strong seedling medium as a whole, place them at 26±2°C, and carry out strong seedling cultivation under the condition of photoperiod of 16h light/8h dark and light intensity of 1500-2000Lux. During the process of strong seedling cultivation The relative humidity is 60-75%. Somatic embryo seedlings are subcultured every 4 weeks during the cultivation of strong seedlings. The roots of somatic embryonic seedlings gradually elongate to form main roots and lateral roots. The embryos develop into stems and leaves. After 2 subcultures, Obtain the strong seedling of cluster seedling test tube (as Fig. 7) with a plurality of stem and leaf ends, continue subculture, have the weak and small stem and leaf end atrophy of the strong seedling of cluster seedling test tube with a plurality of stem and leaf ends, after cultivating 4 weeks, obtain with obvious The test-tube strong seedlings of the dominant trunk (as shown in Figure 8).

7. Hardening, transplanting and planting of test-tube seedlings

Place the culture bottle in a seedling hardening room with shading conditions (50% shading rate) to cultivate for 5-10 days, then open the bottle cap of the culture bottle, and after hardening and cultivating in the transplanting room for 1 day, take out the plant and wash it with tap water The remaining agar medium at the roots of the test-tube seedlings is transplanted into the soilless culture medium of Robinia pseudoacacia (perlite, vermiculite, and the volume ratio of perlite and vermiculite is 1:1) and carries out container culture in the transplanting room, and transplanting culture The temperature is 25±5°C, the relative humidity is 56-90%, and after 7-10 days of cultivation under natural light, the survival rate of transplanting is counted, reaching 100%. The black locust seedlings can be planted in the field 5-6 weeks after transplanting.

Example 2

Except for the callus induction culture process, the 6-BA in the callus culture medium is 0 mg/L, and the 2,4-D is 5.0 mg/L; in the somatic embryo induction culture process, the somatic embryo induction medium The 6-BA used is 1.0 mg/L, the NAA is 1.0 mg/L, the rest is the same as that of Example 1, and the data analysis results are shown in Tables 2 and 3.

Example 3

In addition to the callus induction culture process, the 6-BA in the callus culture medium is 0.3 mg/L, and the 2,4-D is 0.3 mg/L; in the somatic embryo induction culture process, the somatic embryo induction culture The used 6-BA of base is 0.1mg/L, and NAA is 0.1mg/L, and all the other are identical with embodiment 1, and data analysis result is as shown in table 2,4.

Example 4

In addition to the callus induction culture process, the 6-BA in the callus culture medium is 0.5 mg/L, and the 2,4-D is 2.0 mg/L; in the somatic embryo induction culture process, the somatic embryo induction culture The used 6-BA of base is 1.0mg/L, and NAA is 0.5mg/L, and all the other are identical with embodiment 1, and data analysis result is as shown in table 2,4.

Table 4 Somatic Embryo Induction Culture Results

Example 5

Except for the callus induction culture process, the 6-BA in the callus culture medium is 3.0 mg/L, and the 2,4-D is 5.0 mg/L; during the somatic embryo induction culture process, the somatic embryo induction culture The used 6-BA of base is 0mg/L, and NAA is 0.1mg/L, and all the other are identical with embodiment 1, and data analysis result is as shown in table 2,5.

Example 6

Except for the callus induction culture process, the 6-BA in the callus culture medium is 1.0 mg/L, and the 2,4-D is 1.0 mg/L; during the somatic embryo induction culture process, the somatic embryo induction culture The used 6-BA of base is 0.5mg/L, and NAA is 1.0mg/L, and all the other are identical with embodiment 1, and data analysis result is as shown in table 2,5.

Table 5 Results of somatic embryo induction culture

The test results in Tables 2, 3, 4, and 5 show that:

1. The embryonic age of the explant has a significant effect on the callus induction rate (p<0.05), and the immature embryos that are too tender (earlier than 45 days after flowering) or over-ripe (later than 75 days after flowering) cannot obtain ideal Embryogenic callus induction results. The callus rate of immature zygotic embryos collected 55 days after flowering on all induction media was higher than that of explants at other periods, and the induced callus grew fast, was light yellow or light yellow green, and had a compact structure , in granular form, and can still maintain high vigor after many subcultures.

2. In the present invention, MS is used as the basic medium, and 6-BA 0-3.0mg/L, 2,4-D 0.3-5.0mg/L, MES500mg/L, sucrose 30g/L, agar 6g/L are added, The medium with a pH value of 5.8 is a callus induction medium, and the callus induction rate is high, reaching 59.12-95.42%, indicating that exogenous hormones have a significant effect on the average somatic embryo induction number of a single explant (p<0.05) .

3. In the method of the present invention, MS is used as the basic medium, and NAA0.1-2.0mg/L, 6-BA0-1.0mg/L, MES500mg/L, glutamine 250mg/L, hydrolyzed casein 500mg/L are added , sucrose 30g/L, agar 6g/L, and the medium with a pH value of 5.8 is the somatic embryo induction medium, and the induction rate of somatic embryos is high, reaching 50.04%-90.73%, indicating that exogenous hormones have a great influence on the induction of somatic embryos through callus. The effect of somatic embryos was significant (p<0.05).

4. The method of the present invention has a high yield of somatic embryo seedlings obtained by germination of mature somatic embryos, that is, the germination rate of mature somatic embryos is high, reaching more than 80%, and the transplanting survival rate of test-tube plantlets is high, which shows that the method of the present invention The method can form a large number of excellent black locust test-tube seedlings in a short period of time, and can carry out large-scale and factory production.

5. The callus induction rate of explant immature zygotic embryos on the 55th day after flowering in the present invention is 66.72-95.42%, the induction rate of somatic embryos is 76.88-90.73%, and the germination rate of somatic embryos is 80-100% , was significantly higher than that of explants in other periods, and 55 days after flowering was the best period for immature zygotic embryos.

Claims (10)

1. locust tree propagation method comprises as follows step in sequence:

1) gathers the locust tree pod;

2) the locust tree pod is carried out taking out its zygotic embryo behind the surface sterilizing, obtain aseptic zygotic embryo;

3) aseptic zygotic embryo is inoculated on the callus inducing medium, carries out callus induction and cultivate, obtain callus;

4) callus is inoculated on the somatic embryo inducement medium, carries out somatic embryo inducement and cultivate, obtain the proembryo culture;

5) the proembryo culture is inoculated in the maturation of carrying out somatic embryo in the somatic embryo maturation medium and cultivates, obtain somatic embryo;

6) somatic embryo of maturation is inoculated in sprouts cultivation on the germination medium, obtain body embryo seedling;

7) body embryo seedling is inoculated in carries out strong seedling culture on the strong seedling culture base, promote the growth of body embryo seedling, obtain test tube strong sprout;

8) test tube is refined seedling, transplanting strong sprout, promptly get.

2. the method for claim 1; It is characterized in that: described locust tree pod is to be in the locust tree locust tree pod of back in the 25th day to the 75th day this time period of blooming; Be preferably and be in the locust tree locust tree pod of back in the 45th day to the 65th day this time period of blooming; More preferably be in the locust tree locust tree pod of back in the 55th day to the 65th day this time period of blooming, most preferably be locust tree the 55th day the locust tree pod in back of blooming.

3. the method for claim 1; It is characterized in that callus inducing medium described in the step 3) is MS minimal medium+2-(N-morphine quinoline) ethyl sulfonic acid 500mg/L+2; 4-dichlorophenoxyacetic acid 0.3-6.0mg/L+6-benzyl aminoadenine 0-3.0mg/L+ sucrose 30g/L+ agar 6g/L, the pH value is 5.8.

4. like claim 1 or 3 described methods, it is characterized in that callus induction described in the step 3) is cultivated is carrying out under the following condition: under the dark condition, cultivation temperature is 26 ± 2 ℃.

5. the method for claim 1; It is characterized in that the medium of somatic embryo inducement described in the step 4) is MS minimal medium+2-(N-morphine quinoline) ethyl sulfonic acid 500mg/L+ glutamine 250mg/L+ caseinhydrolysate 500mg/L+ methyl 0.1-2.0mg/L+6-benzyl aminoadenine 0-1.0mg/L+ sucrose 30g/L+ agar 6g/L, the pH value is 5.8.

6. the method for claim 1, it is characterized in that somatic embryo inducement described in the step 4) is cultivated is carrying out under the following condition: under the dark condition, cultivation temperature is 26 ± 2 ℃.

7. the method for claim 1 is characterized in that the somatic embryo maturation medium described in the step 5) is MS minimal medium+caseinhydrolysate 500mg/L+ sucrose 30g/L+ agar 6g/L.

8. the method for claim 1 is characterized in that the germination medium described in the step 6) is MS minimal medium+sucrose 30g/L+ agar 6g/L.

9. the method for claim 1 is characterized in that the base of strong seedling culture described in the step 7) is+indolebutyric acid 0.2mg/L+ sucrose 30g/L+ agar 6g/L.

10. the method for claim 1; It is characterized in that institute's strong seedling culture is carried out under following condition in sprouting cultivation described in the ripe cultivation of somatic embryo described in the step 5), the step 6) or the step 7): cultivation temperature is 26 ± 2 ℃; Intensity of illumination is 1500-2000lux, and periodicity of illumination is 14-16 hour illumination/8-10 hour dark; Acclimatization and transplants described in the step 8) comprises as follows step in sequence: open the bottle cap of blake bottle, the refining seedling is 1 day in transplanting the chamber; Then test-tube plantlet is taken out, clean the residual agar medium of root, be transplanted to transplant in the locust tree soilless culture substrate and cultivate 5-6 Zhou Houzai and be colonizated in the land for growing field crops with running water.

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