Culture method for waxberry tissue culture regeneration by taking endosperm as explant

Abstract

According to the waxberry regeneration tissue culture method taking the waxberry endosperm as the explant, the endosperm is taken as the explant, so that the problems that the conventional explant only grows callus, does not grow regeneration buds and is quickly browned are effectively solved, the endosperm is positioned in the seed, direct contact of disinfectant such as alcohol and sodium hypochlorite (NaCLO) is avoided during disinfection, and the explant can be quickly browned. The browning rate is reduced, the activity of polyphenol oxidase (PPO) of endosperm is low, the total phenol content is low, the initial browning degree is natural and light, and the browning rate is lower than that of a traditional method with waxberry leaves and pulp as explants. And performing induced culture under the screened optimal hormone concentration and illumination condition, and performing differentiation culture to generate callus and waxberry regenerated tissue. Compared with the traditional method using leaves or pulp as explants, the browning rate of the method provided by the invention is reduced by 100%, and the generation rate and differentiation rate of calluses are respectively improved by 80%-90% and 2%-4% by virtue of the screened optimal endosperm sampling period and optimal culture environment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of perennial woody plant callus culture and regeneration, and particularly relates to a culture method for bayberry tissue culture regeneration by taking endosperm regeneration cells as explants. BACKGROUND

[0002] Bayberry (Myrica rubra) is an important subtropical fruit crop, is a evergreen fruit tree of Myricaceae Myrica, is a special fruit in south China, is one of the most characteristic and advantageous fruits in Zhejiang Province, and has high nutritional and economic value. At present, bayberry production and cultivation mainly adopts grafting propagation technology. Although the technology can effectively maintain the characteristics of varieties, there are problems of low survival rate and slow propagation speed, which restrict the large-scale development of the bayberry industry. In comparison, the tissue culture technology can reduce production cost and improve propagation efficiency. However, the research and application of the tissue culture of bayberry, especially the regeneration approach of callus, are relatively scarce at present. The main reasons are as follows: it is difficult to sterilize the surface of the explant of bayberry, and Zheng Xiaojun et al. (Preliminary Report on Tissue Culture of Bayberry Stem Segments) pointed out in the research that the contamination rate of bayberry stem segments is extremely high, and needs to be controlled by means of multiple disinfectant alternation treatment and addition of antibiotics. Browing is prone to occur in the process of callus induction, especially the explants such as leaves, stem segments and fruits are rich in various secondary metabolites, and He Xinhua et al. (Research on Browning Problem in Bayberry Tissue Culture) specially studied the browning problem of bayberry tissue culture. Their research confirmed that the activities of polyphenol oxidase (PPO) and peroxidase (POD) in the explants of bayberry are extremely high. These substances significantly inhibit the dedifferentiation of the explants and the formation, growth and subsequent differentiation of callus. Therefore, it is difficult to make substantial breakthroughs in the tissue culture of bayberry, especially the technical system for regenerating complete plants through callus.

[0003] Dongkui bayberry is the best main cultivar in terms of commodity, and the fruit is extra large, high and round, deep red or purple red when ripe, the suture line is obvious, and the fruit stalk is prominent. A transgenic system is an important technology for gene function research, with the rapid development of bayberry research, the market demand for bayberry is gradually increasing, and the plant tissue regeneration is difficult, and the transgenic system cannot be established. Therefore, it is urgent to find a culture method for efficient callus induction and differentiation. SUMMARY

[0004] To solve the above problems, the application provides a culture method for tissue culture regeneration of waxberry by using endosperm as explant. The endosperm of waxberry as explant can effectively solve the problems of only callus growth, no regeneration bud growth and rapid browning of explant. The best growth period of waxberry fruit is selected, the pulp is removed, and the fruit core is smashed after disinfection by alcohol and sodium hypochlorite (NaClO), the unformed endosperm cells are obtained from the inner seed coat, and the waxberry regeneration tissue is obtained after induction culture and differentiation culture. The application provides the endosperm as the explant, which effectively solves the problems of only callus growth, no regeneration bud growth and rapid browning of the conventional explant. Compared with the disinfection of waxberry leaves and pulp, the disinfection is often stimulated by disinfectant liquid to accelerate browning. The endosperm is located in the interior of the seed, and direct contact with disinfectant liquid such as alcohol and NaClO is avoided during disinfection, so that the browning rate is reduced. The polyphenol oxidase (PPO) activity of the endosperm is low, the total phenol content is small, and the initial browning degree is naturally lighter. The browning rate after the traditional use of leaves and pulp as explants is lower. The induction culture and differentiation culture are carried out under the selected optimal hormone concentration and light condition to generate callus and waxberry regeneration tissue. The selected optimal endosperm sampling period and optimal culture environment make the method of the application reduce the browning rate by 100% compared with the traditional method of using leaves or pulp as explants, and the generation rate and differentiation rate of callus are increased by 80%-90% and 2%-4%, respectively.

[0005] Although leaves and pulp are convenient and easy to obtain, and more cases of tissue culture regeneration are carried out by using leaves and pulp as explants, the technology is more mature. However, the waxberry is a perennial woody plant, the degree of lignification of branches is high, the ratio of cutting area / volume is large, a large amount of phenolic substrates is released after cell injury, the PPO activity is high, the quinone polymer is rapidly formed, the browning is fast, the toxicity is heavy, and the bud primordium differentiation is directly inhibited, so the success rate of tissue culture by using the traditional leaves and pulp of waxberry as explants is very low.

[0006] Although the leaves or pulp of waxberry is easy to cause browning and lead to differentiation failure, there is no tissue culture for generating regeneration tissue by using endosperm as explant at present. This is because the endosperm is a tissue existing with embryo development, and the endosperm with regeneration potential must be obtained in a specific and short window period before seed maturation after fertilization. This timing is difficult to accurately grasp, and is limited by the flowering phenology of waxberry. Compared with leaves and stem segments, the endosperm is not as easy to obtain. A large number of studies have proved that the waxberry fruit in the swelling period is selected, a series of treatments are carried out, including removing pulp, alcohol disinfection, sodium hypochlorite soaking, etc., and the unformed endosperm cells wrapped by the inner seed coat can be obtained by smashing the fruit core with a small hammer. The endosperm cells can quickly and safely generate callus and differentiate to generate regeneration tissue under the selected conditions.

[0007] The invention selects the waxberry fruits in the swelling stage, removes the pulp, disinfects with a disinfectant, and is cultured under certain conditions to inhibit browning, induce callus production, and successfully differentiate, and the browning rate is reduced by 100% compared with the traditional browning rate of leaf and pulp as explants.

[0008] In addition, the induction of the initial callus is also a key link, the selection of light conditions and the proportion of hormones in the induction medium will affect the browning, dedifferentiation and callus production of the explants, if the light is too strong, it will promote the original function or differentiation, and will catalyze the oxidation of PPO to generate quinone substances, thereby causing browning. This is not only not conducive to the dedifferentiation of the explants, but also promotes the browning of the explants. By using the method provided by the invention, the light intensity of 0-50PPFD is selected, and the induction medium is used for culture, which can significantly reduce the activity of PPO, inhibit the browning phenomenon, and is conducive to the cell to leave the original differentiation state. Enter the dedifferentiation process, thereby forming callus.

[0009] Finally, the differentiation culture of green callus is also particularly important, the selection of light conditions and the proportion of differentiation medium formula will affect the differentiation of callus, if the light is not enough or too strong, the proportion of hormones is not matched, the callus will continue to proliferate disorderly, and cannot be further differentiated into buds and roots, and finally still changes to browning. By using the method provided by the invention, the light of 0-50PPFD is selected, and the bud medium and root medium are used for culture, which can promote the differentiation of callus to produce regenerative tissue.

[0010] Using the traditional method of leaf and pulp as explants, the browning rate is very high, using the method provided by the invention, using endosperm as explant can not only significantly inhibit browning, but also reduce the browning rate by 100%, and can quickly form callus, and then differentiate to produce regenerative tissue.

[0011] Therefore, the endosperm as an explant for induction culture and differentiation culture provided by the invention to obtain waxberry regenerative tissue has important significance for the establishment of a waxberry transgenic system.

[0012] On the one hand, the invention provides a culture method of waxberry regenerative tissue, mainly by using the endosperm of waxberry fruit as an explant for induction culture and differentiation culture to obtain waxberry regenerative tissue.

[0013] In some ways, the leaves, pulp and endosperm of Myrica rubra are cut as explant materials on the super-clean bench, and are placed in a sterile container containing sterilized water; the explants are immersed in 70-75% alcohol for 25-35 seconds, and are immersed in 2-3% NaClO for 10-15 minutes on the super-clean bench, and are then washed with sterile water for 4-5 times, and are placed in a sterile container, wherein the endosperm cells need to be broken by a small hammer, and after the culture of different explant materials, the leaves and pulp show explant browning phenomenon, which hinders the regeneration of the explants, and the endosperm as the explant avoids the browning phenomenon, and the browning rate is reduced by 100%.

[0014] Further, the endosperm is the endosperm of the swollen Myrica rubra fruit, which is induced to generate callus, and is differentiated to obtain regenerated tissue.

[0015] In some ways, the Myrica rubra fruits in good condition in the hard core period, the swelling period and the mature period are selected, and are divided into three corresponding experimental groups, and are cleaned. The fruits are cut to remove the pulp on the super-clean bench, and are placed in a sterile container containing sterilized water; the explants are immersed in 70-75% alcohol for 25-35 seconds, and are immersed in 2-3% NaClO for 10-15 minutes on the super-clean bench, and are then washed with sterile water for 4-5 times, and are placed in a sterile bag for standby; the fruit core is broken by a small hammer, and the unformed endosperm cells wrapped in the inner integument are placed in the induction medium for culture, and the endosperm in the young fruit period (the hard core period and the swelling period) as the explant has the highest probability of inducing callus, and the endosperm in the mature period as the explant has the highest probability of inducing callus to differentiate into tissue.

[0016] Further, the induction culture adopts a light condition of 0-50 PPFD, and the induction medium is MS medium added with 6-BA 0.1-0.5 mg, 2,4-D 2 mg, sucrose 30 g and agar powder 7 g per liter.

[0017] In some ways, the sterile Myrica rubra endosperm cells are cultured in the initial induction medium under the light conditions of 200 PPFD, 50 PPFD and 0 PPFD, and the temperature is 25℃±2℃; after 15-30 days of culture, a large amount of white callus and a small amount of green callus grow in the 50 PPFD and 0 PPFD environments, that is, the light condition of 0-50 PPFD is beneficial to the growth of callus. In the induction medium, a higher concentration of 2,4-D is a key factor for starting the endosperm cells to separate from the static state and start to divide to form callus, and a lower concentration of 6-BA can avoid early differentiation and thus lead to the early differentiation of callus into adventitious buds, and the differentiation rate of callus in the induction medium can reach 77%.

[0018] Further, the differentiation culture includes bud differentiation culture and root differentiation culture.

[0019] Further, the light condition for the bud differentiation culture is 50PPFD, and the bud culture medium is MS medium added with 6-BA 2 mg, NAA 0.5 mg, sucrose 30 g and agar powder 7 g per liter.

[0020] In some modes, the green callus is transferred to a new differentiation culture medium under the light conditions of 200PPFD, 50PPFD and 0PPFD, and after 15-30d of continuous culture, only the light condition of 0PPFD produces root differentiation, and the differentiation rate is 7%, in the root differentiation culture medium, a high concentration of auxin NAA (1.5-3 mg / L) is dominant to promote cell longitudinal division, induce root primordium formation, and a low concentration of cytokinin 6-BA (1 mg / L) plays a synergistic role, and finally roots are developed.

[0021] Further, the culture method is characterized in that the light condition for the root differentiation culture is 0PPFD, and the root culture medium is MS medium added with 6-BA 1 mg, NAA 1.5-3 mg and sucrose 30 g per liter.

[0022] In some modes, the green callus is transferred to a new differentiation culture medium under the light conditions of 200PPFD, 50PPFD and 0PPFD, and after 15-30d of continuous culture, only the light condition of 0PPFD produces root differentiation, and the differentiation rate is 7%, in the root differentiation culture medium, a high concentration of auxin NAA (1.5-3 mg / L) is dominant to promote cell longitudinal division, induce root primordium formation, and a low concentration of cytokinin 6-BA (1 mg / L) plays a synergistic role, and finally roots are developed.

[0023] In another aspect, the application provides a use of the waxberry fruit endosperm as an explant for the regeneration tissue culture of the waxberry.

[0024] The method for the regeneration tissue culture of the waxberry provided by the application has the following beneficial effects:

[0025] 1. The browning rate is very high when the traditional method is used to take the leaf and pulp as the explant, the browning rate is reduced by 100% when the endosperm is taken as the explant according to the method provided by the application, and the regeneration tissue is quickly formed and then differentiated.

[0026] 2. The de-differentiation is difficult and the callus production rate is low when the traditional method is used to select the improper endosperm period, the de-differentiation success rate is high when the method provided by the application is used to take the swollen period as the explant, and the callus differentiation rate is 80-90%.

[0027] 3, improper culture environment, will prompt the endosperm cells to maintain the original function or differentiation, and will catalyze PPO oxidation to generate quinone substances, thereby triggering browning, if callus is produced, the unsuitable environment will also promote the callus to continue disordered proliferation, and cannot be further differentiated into buds and roots, and eventually still changes to browning. And by using the method provided by the application, selecting the best environmental conditions for culture can significantly reduce the activity of PPO, inhibit the browning phenomenon, and at the same time, it is beneficial for the cells to leave the original differentiation state and enter the dedifferentiation process, thereby forming callus and differentiating to produce regenerated tissue. BRIEF DESCRIPTION OF DRAWINGS

[0028] In Example 1, Figure 1 Leaf (left) and root system (right) produced by differentiation of the endosperm callus of Myrica rubra DETAILED DESCRIPTION

[0029] The preferred embodiments of the application will be further described in conjunction with the accompanying drawings, it should be pointed out that the following embodiments are intended to facilitate the understanding of the application, and do not have any limiting effect on the application, all the features disclosed in the embodiments of the application, or the steps in the disclosed methods or processes, except for the mutually exclusive features and / or steps, can be combined in any way.

[0030] Example 1: Influence of endosperm of Myrica rubra at different stages on callus generation as explant material

[0031] The influence of endosperm at different stages on callus generation as explant material provided by the present embodiment is as follows:

[0032] (1) Selection of explants: select good state hard core period (30-45 days after flowering), swelling period (mid-May to early June), mature period (early June to early July) of Dongkui Myrica rubra fruits, and divide them into corresponding three experimental groups, and clean them.

[0033] (2) Disinfection of explants: remove the pulp of the above fruits on the clean bench, put them into a sterile container containing sterilized water; immerse the explants in 70-75% alcohol on the clean bench for 25-35 seconds, immerse them in 2-3% sodium hypochlorite (NaClO) for 10-15 minutes, then rinse them with sterile water for 4-5 times, and place them in a sterile bag for standby; use a hammer to break the fruit core, and put the unformed endosperm cells wrapped in the inner seed coat into the induction medium.

[0034] (3) Initial callus induction: sterile, as small as possible, broken by forceps, endosperm cells of Myrica rubra were cultured in initial induction medium, the culture condition was light / dark environment, temperature 25℃±2℃; after 15-30d, a large number of white and a small number of green calli grew out, the initial induction medium was MS medium added with 6-BA 0.1-0.5mg, 2,4-D 2mg, sucrose 30g, agar powder 7g per liter.

[0035] (4) Differentiation culture: the intact green callus was transferred to new differentiation medium and cultured for 15-30d until new shoots or roots grew out, such as Figure 1 , the shoot culture condition was light intensity 50PPFD, photoperiod 10-14h / d, the root culture condition was dark condition, temperature 25℃±2℃; the root differentiation medium was MS medium added with 6-BA 1mg, NAA 1.5-3mg, sucrose 30g per liter; the shoot differentiation medium was MS medium added with 6-BA 2mg, NAA 0.5mg, sucrose 30g, agar powder 7g per liter.

[0036] The MS medium contained per liter: macronutrients: potassium nitrate KNO3 1900mg, ammonium nitrate NH4NO3 1650mg, potassium dihydrogen phosphate KH2PO3 170mg, magnesium sulfate MgSO4·7H2O 370mg; calcium salt: calcium chloride CaCl2·2H2O 440mg; micronutrients: potassium iodide KI 0.83mg, boric acid H3BO3 6.2mg, manganese sulfate MnSO4·4H2O 22.3mg, zinc sulfate ZnSO4·7H2O 8.6mg, sodium molybdate Na2MoO4·2H2O 0.25mg, copper sulfate CuSO4·5H2O 0.025mg, cobalt chloride CoCl2·6H2O 0.025mg; iron salt: disodium ethylenediaminetetraacetate Na2-EDTA 37.25mg, ferrous sulfate FeSO4·7H2O 27.85mg; sucrose 30g, agar powder 7g, pH 5.7;

[0037] The number of calli generated and the differentiation rate of the three groups were investigated, and the results were shown in Table 1:

[0038] Table 1 Influence of endosperm of Myrica rubra at different periods as explant material on callus generation

[0039] Explant type Explant number Callus differentiation number Callus differentiation rate Browning number Differentiation number Differentiation rate Hard core endosperm 50 45 90％ 0 2 4％ Swelling endosperm 50 40 80％ 0 1 2％ Mature endosperm 50 15 30％ 0 3 6％

[0040] As can be seen from Table 1, the endosperm in the young fruit stage (hard core stage and swelling stage) has the highest probability of inducing callus, reaching 80%-90%, the endosperm in the mature stage has a lower probability of inducing callus than the other two stages, being 30%, the endosperm in the three stages does not brown, the browning rate is 0, the endosperm in the mature stage has the highest probability of differentiating into tissue, being 6%, the endosperm in the hard core stage and the swelling stage has a probability of differentiating into tissue of 4% and 2% respectively. Since the endosperm in the hard core stage is not shaped, the amount is small, and it is not easy to obtain, therefore, the endosperm in the swelling stage is better as an explant.

[0041] Example 2 Influence of different light intensities and hormone concentrations on callus generation

[0042] The influence of different light intensities and hormone concentrations on callus generation provided by the present embodiment is as follows:

[0043] (1) Selection of explants: select well-conditioned swelling stage Myrica rubra fruits, and clean them.

[0044] (2) Disinfection of explants: remove the pulp of the above fruits on the clean bench, and place them in a sterile container containing sterilized water; immerse the explants in 70-75% alcohol for 25-35 seconds for disinfection on the clean bench, immerse them in 2-3% sodium hypochlorite (NaClO) for 10-15 minutes for treatment, then rinse them with sterile water for 4-5 times, and place them in a sterile bag for standby; use a small hammer to break the fruit core, and place the unformed endosperm cells wrapped in the inner seed coat into the induction medium;

[0045] (3) Initial callus induction: culture the sterile Myrica rubra endosperm cells in the initial induction medium, and divide them into two groups, one group sets different light intensities, i.e. culture conditions are strong light (200 PPFD) / weak light (50 PPFD) / dark environment (0 PPFD), temperature is 25℃±2℃; the induction medium is consistent, and after 15-30 days of culture, the number and differentiation rate of callus differentiation under the three light conditions are examined, and the results are shown in Table 2; one group sets different hormone concentrations of the medium, i.e. 2mg / L 2,4-D is respectively matched with 0.1mg / L 6-BA, 0.5mg / L 6-BA, 1mg / L 6-BA, 2mg / L 6-BA, and 0.5mg / L 6-BA is respectively matched with 0.5mg / L 2,4-D, 1mg / L 2,4-D, 2mg / L 2,4-D, 3mg / L 2,4-D as the induction medium for callus culture, and the number and differentiation rate of callus differentiation are examined, and the results are shown in Table 3:

[0046] Table 2 Influence of different light intensities on callus generation

[0047] Light intensity Explant number Callus differentiation number Callus differentiation rate Browning number Differentiation number Differentiation rate 0 PPFD 50 42 84％ 0 2 4％ 50 PPFD 50 40 80％ 0 1 2％ 200 PPFD 50 45 90％ 0 0 0％

[0048] As shown in Table 2, weak light and dark environment is conducive to the growth of callus, and the differentiation rate of strong light is the lowest, the number of differentiation is 0, and the process of dedifferentiation and callus generation is inhibited.

[0049] Table 3 Effect of different hormone concentrations on callus generation

[0050]

[0051] As shown in Table 3, the number of callus differentiation is the largest when the concentration of two kinds of hormones is 2,4-D 2mg / L and 6-BA 0.1-0.5mg / L, and the differentiation rate is 3.3% and 6.7% respectively, so the optimal formula of the initial induction medium is: adding 6-BA 0.1-0.5mg, 2,4-D 2mg, sucrose 30g, agar powder 7g per liter of MS medium.

[0052] Example 3 Effect of different light intensities and hormone concentrations on root and bud differentiation

[0053] The effect of different light intensities and hormone concentrations on callus generation provided in this embodiment is as follows:

[0054] (1) Selection of explants: select well-developed Dongkui Yangmei fruits, and clean them.

[0055] (2) Disinfection of explants: remove the pulp of the fruits in the above-mentioned step on the clean bench, and put them into a sterile container containing sterilized water; immerse the explants in 70-75% alcohol for 25-35 seconds and 2-3% sodium hypochlorite (NaClO) for 10-15 minutes on the clean bench, then rinse them with sterile water for 4-5 times, and place them in a sterile bag for standby; use a hammer to break the fruit core, and put the unformed endosperm cells wrapped in the inner seed coat into the induction medium;

[0056] (3) Initial callus induction: culture the sterile endosperm cells of Yangmei in the initial induction medium, and the culture conditions are light / dark environment and temperature 25℃±2℃; after 15-30 days of culture, a large number of white and a small number of green calli are grown, and the initial induction medium is: adding 6-BA 0.1-0.5mg, 2,4-D 2mg, sucrose 30g, and agar powder 7g per liter of MS medium.

[0057] (4) Tissue differentiation culture: the green callus was divided into two groups and transferred to new differentiation medium, one group was set to different light intensity, i.e. culture conditions were strong light (200 PPFD) / weak light (50 PPFD) / dark environment (0 PPFD), photoperiod 10-14 h / d, temperature 25℃±2℃; the differentiation medium was consistent, and the number of root differentiation and the number of bud differentiation under three light conditions were examined after 15-30d of culture, and the results are shown in Table 4; one group was set to different hormone concentration medium, i.e. 2mg / L NAA was respectively matched with 0.5mg / L 6-BA, 1mg / L 6-BA, 1.5mg / L 6-BA, 2mg / L 6-BA, 3mg / L 6-BA, and 2mg / L 6-BA was respectively matched with 0.5mg / L NAA, 1mg / L NAA, 1.5mg / L NAA, 2mg / L NAA, 3mg / L NAA as differentiation medium, and the number of root differentiation and the number of bud differentiation were examined after 15-30d of culture until new shoots or roots grew out, and the results are shown in Table 5:

[0058] Table 4 Influence of different light intensities on root and bud differentiation

[0059] Light intensity Callus number Root differentiation number Shoot differentiation number 0 PPFD 30 2 0 50 PPFD 30 0 1 200 PPFD 30 0 0

[0060] As shown in Table 4, dark environment is conducive to root differentiation, and weak light is conducive to bud differentiation, the number of root differentiation is 2 in dark environment, and the number of bud differentiation is 1 in weak light environment.

[0061] Table 5 Influence of different hormone concentrations in medium on root and bud differentiation

[0062]

[0063] As shown in Table 5, the medium with the highest number of root differentiation has a concentration of 6-BA 1mg / L and NAA 1.5-3mg / L, and the number of differentiation is 1, and the medium with the highest number of bud differentiation has a concentration of 6-BA 2mg / L and NAA 0.5mg / L, therefore, the root medium is MS medium with 6-BA 1mg, NAA 1.5-3mg, sucrose 30g per liter, and the optimal formula of bud medium is MS medium with 6-BA 2mg, NAA 0.5mg, sucrose 30g, and agar powder 7g per liter.

[0064] Example 4 Influence of different parts of Myrica rubra as explant material on callus generation

[0065] The influence of different parts as explant material on callus generation provided in this example is as follows:

[0066] (1) Cut bayberry leaves, pulp and endosperm at different stages as explant materials in a laminar flow hood and place them in a sterile container filled with sterile water. Soak the explants in 70-75% alcohol for 25-35 seconds and 2-3% sodium hypochlorite (NaClO) for 10-15 minutes. Then rinse them with sterile water 4-5 times and place them in a sterile container. The endosperm cells need to be crushed with a small hammer.

[0067] (2) Initial callus induction: Treated sterile explants were cultured in an initial induction medium under light / dark conditions at 25℃±2℃. After 15-30 days of culture, the initial induction medium was MS medium supplemented with 0.1-0.5 mg 6-BA, 2 mg 2,4-D, 30 g sucrose, and 7 g agar powder per liter. The number and differentiation rate of callus formation were examined, and the results are shown in Table 6.

[0068] Table 6. Effects of different parts of Myrica rubra as explant materials on callus formation.

[0069] Explant type Explant number Callus differentiation number Browning number Differentiation number Differentiation rate Leaf 30 0 30 0 0％ Hard core pulp 30 0 30 0 0％ Swelling pulp 30 0 30 0 0％ Mature pulp 30 0 30 0 0％ Hard core endosperm 50 45 0 2 4％ Swelling endosperm 50 40 0 1 2％ Mature endosperm 50 15 0 3 6％

[0070] As shown in Table 6, both leaves and pulp exhibited explant browning, which hindered explant regeneration. Endosperm, as an explant, did not show browning. Therefore, using endosperm as an explant for callus differentiation and regeneration of tissue yielded the best results.

[0071] The application of this invention is not limited thereto. It can be expanded according to its application in plant callus culture and regeneration technology. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of this invention; therefore, the scope of protection of this invention should be determined by the scope defined in the claims.

Claims

1. A method for culturing regenerated tissue of *Myrica rubra*, characterized in that, This includes using the endosperm of bayberry fruit as an explant for induction and differentiation culture to obtain bayberry regeneration tissue.

2. The cultivation method as described in claim 1, characterized in that, The endosperm is the endosperm of bayberry fruit during the expansion stage, which is induced to produce callus tissue and then redifferentiated to obtain regenerated tissue.

3. The cultivation method as described in claim 2, characterized in that, The induction culture was conducted under light conditions of 0-50 PPFD, and the induction medium was MS medium supplemented with 0.1-0.5 mg / L of 6-benzylaminopurine (6-BA), 2-3 mg / L of 2,4-dichlorophenoxyacetic acid (2,4-D), 25-35 g / L of sucrose, and 6-8 g / L of agar powder.

4. The cultivation method according to any one of claims 1 to 3, characterized in that, The differentiation culture includes shoot differentiation culture and root differentiation culture.

5. The cultivation method as described in claim 4, characterized in that, The bud differentiation culture was conducted under light conditions of 50-60 PPFD, and the bud culture medium was MS medium supplemented with 2-3 mg / L 6-BA, 0.3-0.5 mg / L naphthaleneacetic acid (NAA), 25-35 g / L sucrose, and 6-8 g / L agar powder.

6. The cultivation method as described in claim 4, characterized in that, The root differentiation culture was conducted under light conditions of 0-20 PPFD, and the root culture medium was MS medium supplemented with 1-1.5 mg / L 6-BA, 1.5-3 mg / L NAA, and 25-35 g / L sucrose.

7. A type of bayberry regeneration tissue, characterized in that, Prepared using the method described in any one of claims 1 to 7.

8. A callus differentiation and regeneration system for *Myrica rubra*, characterized in that, This includes the endosperm of bayberry fruit, induction medium, and differentiation medium.

9. The regeneration system as described in claim 8, characterized in that, The endosperm was the endosperm of the waxberry fruit during its expansion stage. The induction medium was MS medium supplemented with 0.1-0.5 mg / L 6-BA, 2-3 mg / L 2,4-D, 25-35 g / L sucrose, and 6-8 g / L agar powder. The differentiation medium included bud medium and root medium. The bud medium was MS medium supplemented with 2-3 mg / L 6-BA, 0.3-0.5 mg / L NAA, 25-35 g / L sucrose, and 6-8 g / L agar powder. The root medium was MS medium supplemented with 1-2 mg / L 6-BA, 1.5-3 mg / L NAA, and 25-35 g / L sucrose.

10. The use of the endosperm of bayberry fruit as a reagent for constructing a callus differentiation and regeneration system of bayberry.

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