Litsea cubeba secondary embryo proliferation culture medium and proliferation culture method

Abstract

The invention discloses a litsea cubeba secondary embryo proliferation culture medium and a proliferation culture method, the culture medium is based on an MS culture medium, cane sugar, agar, casein hydrolysate, 6-benayl aminopurine, naphthylacetic acid, inositol and cyclodextrin are added, and the pH value is 5.6-5.8; the method comprises the following steps: a somatic embryo generation stage, a secondary embryo proliferation stage and regular removal of mature somatic embryos. According to the method, the immature seeds are adopted as explants, through the somatic embryogenesis culture medium and the secondary embryo proliferation culture medium of a specific formula, dark culture and periodic subculture are combined, the proliferation efficiency of the secondary embryos is remarkably improved, the highest proliferation multiple can reach 20.17 times, meanwhile, the endophyte pollution risk is reduced, and the genetic stability is guaranteed. The method is simple and convenient to operate, short in culture period, low in cost and suitable for large-scale industrial application, provides important technical support for rapid propagation, germplasm preservation and industrial development of litsea cubeba, and has remarkable economic and social benefits.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant tissue culture, and relates to a Litsea cubeba secondary embryo proliferation culture medium and a proliferation culture method thereof. Background Art

[0002] The fruits of Litsea cubeba are rich in citral (60%-90%). The fruits and seeds of Litsea cubeba can not only be used to extract spices, but also have various medicinal values such as antibacterial and anti-inflammatory effects, and it has extremely high development value in the fields of spices, food, medicine and biological control. However, at present, there are still many problems in the industrial breeding technology of Litsea cubeba, which seriously restricts the popularization of improved varieties and large-scale cultivation.

[0003] At present, the seedling breeding technologies of Litsea cubeba mainly include four methods: seed propagation, cuttage propagation, grafting propagation and tissue culture. These traditional breeding methods all have the following problems:

[0004] Seed propagation: The seed germination rate is low, the economic cost is high, and the offspring character segregation is serious, making it difficult to maintain the excellent characters of the parent, and it is not suitable for large-scale afforestation or industrial development.

[0005] Cuttage propagation: Since the wound of Litsea cubeba branches is extremely easy to oxidize and is restricted by natural factors, the propagation coefficient of cuttage propagation is relatively low.

[0006] Grafting propagation: Similarly restricted by natural factors, the propagation coefficient is relatively low, and the gender of the seedlings is uncontrollable.

[0007] Tissue culture: The existing tissue culture methods mainly use branches as explants, and there are problems such as high pollution rate (endophytic bacteria are difficult to kill), low proliferation rate, and difficult differentiation. According to the applicant's previous research, using the seed embryo as an explant for tissue culture, a stage result of an induction rate of 17.5% of embryogenic callus was obtained. During the continuous research process, it was found that due to the restriction of the embryogenic efficiency of callus, the somatic embryo incidence rate is still relatively low, which directly affects the efficiency of plant regeneration, and there is still a bottleneck in large-scale production and application that needs to be broken through. Summary of the Invention

[0008] Aiming at the deficiencies of the existing technology, especially the problems of low induction efficiency of embryogenic callus and low embryogenic efficiency of callus, the purpose of the present invention is to provide an efficient Litsea cubeba secondary embryo proliferation culture medium and a proliferation culture method thereof, so as to improve the breeding efficiency of Litsea cubeba and provide technical support for the large-scale cultivation and industrial development of Litsea cubeba.

[0009] Through long-term exploration and attempts, as well as multiple experiments and efforts, and continuous reform and innovation, in order to solve the above technical problems, the technical solution provided by the present invention is to provide a Litsea cubeba secondary embryo proliferation culture medium, and the medium formula is as follows:

[0010] Based on MS medium, add 30 g / L sucrose, 7 g / L agar, 500 mg / L casein hydrolysate, 0.1 mg / L 6-benzylaminopurine, 0.5 mg / L naphthaleneacetic acid, 600 mg / L inositol, 200 mg / L cyclodextrin, and the pH value is 5.6 - 5.8.

[0011] The said medium is used for the proliferation culture of the secondary embryos of Litsea cubeba.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] By optimizing the medium formula, the present invention significantly improves the proliferation efficiency of secondary embryos. Compared with the prior art, the medium of the present invention is based on MS medium and adds specific concentrations of sucrose, agar, casein hydrolysate, 6-benzylaminopurine, naphthaleneacetic acid, inositol, and cyclodextrin, and can achieve a secondary embryo proliferation multiple as high as 20.17 times within 30 days, and the proliferation efficiency is significantly higher than the traditional method.

[0014] The present invention also provides a method for the proliferation culture of secondary embryos of Litsea cubeba, which includes the following steps:

[0015] (a) Somatic embryo genesis stage: Disinfect the explants of immature seeds of Litsea cubeba and inoculate them into the somatic embryo genesis medium, and culture them in the dark to induce the formation of embryogenic callus and somatic embryos;

[0016] (b) Secondary embryo proliferation stage: Select somatic embryos at the globular embryo stage and inoculate them into the secondary embryo proliferation medium described in claim 3, and culture them in the dark to complete the proliferation of secondary embryos;

[0017] (c) Regularly remove mature somatic embryos and conduct subculture once according to a preset period to maintain the continuous proliferation activity of embryogenic cells.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The present invention uses immature seeds as explants, and through the somatic embryo genesis medium and secondary embryo proliferation medium with specific formulas, combined with dark culture and regular subculture, significantly improves the proliferation efficiency of secondary embryos, with the highest proliferation multiple up to 20.17 times. At the same time, it reduces the risk of endophyte contamination and ensures genetic stability. This method is easy to operate, has a short culture period, and low cost, and is suitable for large-scale industrial application, providing important technical support for the rapid propagation, germplasm preservation, and industrial development of Litsea cubeba, and having significant economic and social benefits.

[0020] On the basis of the above technical solutions, the present invention can also be improved as follows:

[0021] Furthermore, the somatic embryogenesis medium is based on MS medium, supplemented with 30 g / L sucrose, 7 g / L agar, 700 mg / L casein hydrolysate, 2.0 mg / L 6-benzylaminopurine, 0.6 mg / L 2,4-dichlorophenoxyacetic acid, 0.5 mg / L indoleacetic acid, and the pH value is 5.8.

[0022] Compared with the prior art, the beneficial effects of adopting the above further technical solution are as follows:

[0023] By precisely regulating the components of the medium and the concentration of plant growth regulators, the somatic embryogenesis efficiency of Litsea cubeba has been significantly improved, and the highest somatic embryo incidence rate reaches 23.33%.

[0024] On the basis of the above technical solution, the present invention can be further improved as follows:

[0025] Furthermore, in the step (a), the immature seeds are collected in mid-June, and the disinfection steps include rinsing with running water and soaking in 75% alcohol.

[0026] Preferably, the disinfection steps are specifically rinsing with running water for 1 h and soaking in 75% alcohol for 3 - 5 min.

[0027] Compared with the prior art, the beneficial effects of adopting the above further technical solution are as follows:

[0028] Compared with the prior art, by further optimizing the collection time of immature seeds and the disinfection steps, the activity of embryonic cells of the seeds can be ensured, the risk of endophyte contamination can be reduced, the somatic embryo incidence rate and the subsequent secondary embryo proliferation efficiency can be improved, providing a reliable guarantee for the efficient tissue culture of Litsea cubeba.

[0029] On the basis of the above technical solution, the present invention can be further improved as follows:

[0030] Furthermore, in the step (a), after dark culture for 90 days, the incidence rate of cellular embryos reaches 23.3%.

[0031] Compared with the prior art, the beneficial effects of adopting the above further technical solution are as follows:

[0032] By optimizing the dark culture time and somatic embryogenesis conditions, the present invention increases the somatic embryo incidence rate to 23.3%, breaks through the restriction of the embryogenic callus embryogenic efficiency in the prior art, significantly improves the somatic embryogenesis efficiency of Litsea cubeba, provides a higher basic success rate for subsequent secondary embryo proliferation and plant regeneration, and effectively solves the problem that the plant regeneration efficiency is limited due to the low somatic embryo incidence rate in the prior art.

[0033] On the basis of the above technical solution, the present invention can be further improved as follows:

[0034] Furthermore: In the step (b), the inoculation amount of the globular embryos is 0.2 - 0.4 g / dish, and the culture temperature is 24 ± 2 °C.

[0035] Compared with the prior art, the beneficial effects of adopting the above further technical solution are as follows:

[0036] By optimizing the inoculation amount and culture temperature of the globular embryos, the present invention ensures the efficient proliferation of secondary embryos under suitable nutritional and environmental conditions. The proliferation multiple can reach up to 20.17 times at most, significantly improving the proliferation efficiency and quality of secondary embryos, and providing reliable technical support for the large-scale propagation of Litsea cubeba.

[0037] On the basis of the above technical solution, the present invention can also be improved as follows:

[0038] Furthermore: In the step (c), the subculture period is 30 days. Calculated by the fresh weight ratio, the proliferation multiple of secondary embryos reaches 20.17 times.

[0039] Compared with the prior art, the beneficial effects of adopting the above further technical solution are as follows:

[0040] The proliferation multiple of secondary embryos reaches 20.17 times, significantly improving the proliferation efficiency of secondary embryos, breaking through the bottleneck of low proliferation efficiency in the prior art, and providing efficient and stable technical support for the large-scale propagation of Litsea cubeba. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0042] Figure 1 It is a morphological observation diagram of the indirect somatic embryo formation pathway of Litsea cubeba seeds in a preferred embodiment of Method 1 of the present invention. Figure 1 In it, a is a clump-like embryogenic callus, b is a yellow clump-like embryogenic callus, c is a yellow granular embryogenic callus, d is a white granular embryogenic callus, e is a globular embryo, f is a heart-shaped embryo, g is a cotyledon embryo, and h is a secondary embryo proliferation diagram.

[0043] Figure 2 It is a morphological observation diagram of the direct somatic embryo formation pathway of Litsea cubeba seeds in a preferred embodiment of Method 1 of the present invention. Figure 2 In it, both a and b are primary embryos directly formed from Litsea cubeba seeds.

[0044] Figure 3 It is a non-embryogenic cotyledon-like structure.

[0045] Figure 4 It is a diagram for observing the process of secondary embryo proliferation in a preferred embodiment of the method of the present invention. Figure 4 In it, a is the morphological observation diagram on the 0th day of the experimental treatment, b is the morphological observation diagram on the 21st day, c is the morphological observation diagram on the 30th day, and d is the morphological observation diagram on the 21st day after culturing after removing the secondary embryos.

[0046] Figure 5 It is the blank control group for secondary embryo proliferation. Figure 5 In it, a is the morphological observation diagram on the 0th day of culturing in the control group, and b is the morphological observation diagram on the 30th day of culturing. Detailed implementation manners

[0047] The following is an illustration in conjunction with specific embodiments.

[0048] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments in the present invention is not intended to limit the scope of the claimed present invention, but merely represents selected embodiments of the present invention.

[0049] In the present invention, unless otherwise specified, all devices and raw materials can be purchased from the market or are commonly used in this industry. The methods in the following embodiments, unless otherwise specified, are conventional methods in this field.

[0050] Example 1

[0051] In this example, the culture medium formulations for two stages are described:

[0052] (a) Somatic embryo induction medium: Based on the MS medium, adding 30 g / L sucrose, 7 g / L agar, 700 mg / L casein hydrolysate (Ch), 2.0 mg / L 6-benzylaminopurine (6-BA), 0.6 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg / L indoleacetic acid (IAA), and the pH value is 5.8;

[0053] (b) Secondary embryo proliferation medium: Based on the MS medium, adding 30 g / L sucrose, 7 g / L agar, 500 mg / L casein hydrolysate (Ch), 0.1 mg / L 6-benzylaminopurine (6-BA), 0.5 mg / L naphthaleneacetic acid (NAA), 600 mg / L inositol, 200 mg / L cyclodextrin, and the pH value is 5.6 - 5.8.

[0054] Example 2

[0055] This example describes the detailed steps of the proliferation culture method of Litsea cubeba secondary embryos, and the specific steps are as follows:

[0056] 1. Somatic embryo formation stage

[0057] In the somatic embryo formation stage, immature seeds of Litsea cubeba are collected first. Seeds in mid-June are selected to ensure the activity of embryogenic cells. After seed collection, the fleshy pericarp is removed, and the seeds are rinsed under running water with dishwashing liquid for 1 hour to remove surface impurities. Subsequently, they are disinfected by soaking in 75% alcohol for 5 minutes on a sterile operating table, and finally the fruit shells (inner pericarp) are peeled off to obtain seed explants. The disinfected seed explants are inoculated into the somatic embryo formation medium. The medium formula is: based on MS medium, adding 30 g / L sucrose, 7 g / L agar, 700 mg / L casein hydrolysate, 2.0 mg / L 6-benzylaminopurine, 0.6 mg / L 2,4-dichlorophenoxyacetic acid, 0.5 mg / L indoleacetic acid, and the pH value is 5.8. The culture condition is dark culture, the temperature is 24±2°C, and the culture time is 90 days. By optimizing the culture conditions, the somatic embryo incidence rate reaches 23.33%, significantly improving the somatic embryo formation efficiency of Litsea cubeba.

[0058] 2. Secondary embryo proliferation stage

[0059] In the secondary embryo proliferation stage, somatic embryos at the globular embryo stage of development are selected, and about 0.3 g of globular embryos are picked up with a small spoon and inoculated into the secondary embryo proliferation medium. The medium formula is: based on MS medium, adding 30 g / L sucrose, 7 g / L agar, 500 mg / L casein hydrolysate, 0.1 mg / L 6-benzylaminopurine, 0.5 mg / L naphthaleneacetic acid, 600 mg / L inositol, 200 mg / L cyclodextrin, and the pH value is 5.6 - 5.8. The culture condition is dark culture, the temperature is 24±2°C, and the culture time is 30 days. By optimizing the medium formula and culture conditions, the highest secondary embryo proliferation multiple can reach 20.17 times, significantly improving the secondary embryo proliferation efficiency.

[0060] 3. Regularly remove mature somatic embryos

[0061] During the secondary embryo proliferation process, regularly removing mature somatic embryos is a key step to maintain the continuous proliferation of embryogenic cells. The specific operation is as follows: After 30 days of culture, observe the maturity of somatic embryos and timely remove the mature somatic embryos. After removing the mature somatic embryos, the remaining embryogenic cells in the dish will continue to proliferate, thus realizing continuous secondary embryo proliferation. Subculture is carried out every 30 days to maintain the activity of embryogenic cells. By regularly removing mature somatic embryos and subculturing, the problems of somatic embryo aging and insufficient nutrient supply can be effectively avoided, ensuring the efficient proliferation of secondary embryos.

[0062] Example 3

[0063] To further illustrate the technical effects of the present invention and the importance of the setting of each technical detail, the following is illustrated by specific experimental data.

[0064] 1. Effects of different hormone combinations on somatic embryogenesis

[0065] On June 15, the seeds of Litsea cubeba in the traditional Chinese medicine garden of Jiangxi Agricultural University were collected as materials. After removing the fleshy pericarp, dishwashing liquid was added and rinsed under running water for 1 h. Then, it was disinfected with 75% alcohol for 5 min on the sterile operating table, and the fruit shell (endocarp) was peeled off and inoculated on MS + 30 g / L sucrose + 7.5 g / L agar powder + 700 mg / L Ch (casein hydrolysate).

[0066] The experiment selected different concentrations of cytokinin 6-BA (0.5 mg / L, 1.0 mg / L, 1.5 mg / L, 2.0 mg / L), combined with auxins IAA (0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L), 2,4-D (0.25 mg / L, 0.5 mg / L, 0.75 mg / L, 1 mg / L) (Table 1), and designed an orthogonal experiment with three factors and four levels to analyze the effects of different combinations of exogenous hormones on somatic embryogenesis of seeds.

[0067] In this experiment, 10 Litsea cubeba seeds were inoculated in each dish, and each treatment was repeated 3 times. The number of explants in each experimental treatment was not less than 30. All cultures were placed in an environment of 24 ± 2 °C and dark-cultured for 3 months.

[0068] Somatic embryo incidence rate = (number of direct + indirect occurrences) / number of inoculations

[0069] Table 1 Hormones on embryogenic callus induction of Litsea cubeba L 16 (4 3 ) Orthogonal experimental design

[0070]

[0071] After 3 months of inoculation, the somatic embryo incidence rates under different hormone combination treatments are shown in Table 2. It can be seen from Table 2 that somatic embryogenesis was observed in CK (control group), treatments 1, 2, 3, 5, 8, 12, 14, and 15 in the experiment, and the somatic embryo incidence rate of treatment 15 was the highest, reaching 23.3%. No somatic embryogenesis was observed in the remaining treatments.

[0072] For L 16 (4 3)Range analysis was performed on the somatic embryo incidence results of the orthogonal experiment. The results showed that without considering the interaction, the primary and secondary order of the factors was IAA > 6-BA > 2,4-D, and the optimal combination for somatic embryo formation was A4B3C2, that is, 2.0 mg / L 6-BA + 0.6 mg / L 2,4-D + 0.5 mg / L IAA.

[0073] Table 2 Hormones on the Somatic Embryo Genesis of Litsea cubeba L 16 (4 3 )Orthogonal Experiment Results

[0074]

[0075]

[0076] In this example, the somatic embryo formation pathway includes an indirect formation pathway ( Figure 1 ) and a direct formation pathway ( Figure 2 ).

[0077] Figure 1 The indirect pathway of somatic embryo formation in Litsea cubeba is shown, where:

[0078] a. Massive embryogenic callus: The explant forms massive embryogenic callus in the induction medium.

[0079] b. Yellow massive embryogenic callus: The embryogenic callus gradually turns yellow, indicating that it begins to differentiate.

[0080] c. Yellow granular embryogenic callus: The embryogenic callus further differentiates into a granular structure.

[0081] d. White granular embryogenic callus: The embryogenic callus turns white, and the granular structure becomes more obvious.

[0082] e. Globular embryo: The embryogenic callus differentiates into a globular embryo.

[0083] f. Heart-shaped embryo: The globular embryo further differentiates into a heart-shaped embryo.

[0084] g. Cotyledon embryo: The heart-shaped embryo further differentiates into a cotyledon embryo.

[0085] h. Secondary embryo proliferation: The cotyledon embryo begins to proliferate to form secondary embryos.

[0086] Figure 2 The direct formation pathway of somatic embryo formation in Litsea cubeba is shown, where:

[0087] a. The explant directly differentiates into a primary embryo.

[0088] b. The primary embryo further develops, and its morphology gradually becomes clear.

[0089] Figure 3 shows the non-embryogenic material - cotyledonary structures obtained during the completion of the present invention, wherein,

[0090] a. Only cotyledonary structures that cannot proliferate appear,

[0091] b. Cotyledonary structures that cannot proliferate and non-embryogenic callus appear.

[0092] Non-embryogenic materials cannot be further differentiated into somatic embryos, and their morphology is easily confused with cotyledon embryos. In plant tissue culture, differentiating embryogenic and non-embryogenic materials is crucial for optimizing culture conditions and improving the efficiency of somatic embryogenesis.

[0093] Callus is divided into embryogenic callus and non-embryogenic callus. Embryogenic callus is the basis for somatic embryogenesis. Only embryogenic callus can be further differentiated into somatic embryos. The somatic embryo induction rate is a key indicator to measure the efficiency of somatic embryogenesis, which directly determines the potential of secondary embryo proliferation. Only embryogenic callus has sufficient embryogenicity and can efficiently form somatic embryos under suitable culture medium components and culture conditions.

[0094] In this example, the somatic embryo induction rate reaches 23.33%, and the actual application effect in production is significantly higher than the embryogenic callus induction rate of 17.5% in previous studies, indicating that this example more efficiently realizes the transformation from callus to somatic embryos and has an actual impact on large-scale production.

[0095] The induction rate of cotyledonary structures is negatively correlated with the induction rate of embryogenic callus and the somatic embryo induction rate. The formation of cotyledonary structures is due to inappropriate culture conditions (such as hormone concentration, culture time, etc.), resulting in the callus deviating from the embryogenic direction. A higher induction rate of cotyledonary structures will reduce the somatic embryo induction rate because the embryogenic ability of the callus is weakened.

[0096] Experiments show that the culture medium formula and explants used in this example are more conducive to the direct generation of somatic embryos and have substantial technical advantages for the proliferation of secondary embryos.

[0097] 2. Proliferation of secondary embryos

[0098] Directly generated somatic embryos can induce embryogenic callus for the proliferation of secondary embryos, but the proliferation rate is lower than that of indirectly generated somatic embryos.

[0099] In this example, small granular Litsea cubeba spherical embryos with consistent size and good growth induced are selected ( Figure 1(in the medium figure), use a small spoon to pick about 0.3 g and evenly inoculate it into the proliferation medium: MS + 30 g / L sucrose + 7.5 g / L agar + 500 mg / L Ch. Different concentrations of 6-BA (0.1, 0.3, 0.5 mg / L), NAA (0.2, 0.5, 0.8 mg / L), inositol (200, 400, 600 mg / L), and cyclodextrin (100, 200, 300 mg / L) are added to the medium. Set up a four-factor and three-level experiment (Table 3). This experiment has a total of 9 treatment groups + 1 control group (CK). 6 plates are inoculated for each treatment, and every two plates are used as a group of experiments, with three repetitions.

[0100] All cultures are placed in an environment of 24 ± 2 °C and cultured in the dark;

[0101] Observe and record the secondary embryo proliferation and somatic embryo induction. After the proliferation culture is completed, use an analytical balance with a precision of 0.001 to weigh the weight of the cell mass in each plate of the culture, and finally calculate the proliferation multiple of each plate.

[0102] At the same time, continuously observe the differentiation process of embryogenic callus under a stereomicroscope to determine the more suitable proliferation subculture cycle and somatic embryo induction cycle.

[0103] Secondary embryo proliferation multiple = (fresh weight of secondary embryos after one month - fresh weight at inoculation) / fresh weight at inoculation

[0104] Table 3 Effects of hormones and organic additives on the proliferation of secondary embryos of Litsea cubeba L9(3 4 ) Orthogonal experimental design

[0105]

[0106] After the proliferation culture is completed, weigh the quality before and after the experiment to obtain the proliferation multiple. As can be seen from Table 4, a large amount of secondary embryo proliferation can be achieved in Treatment 1, Treatment 6, and Treatment 8. Among them, in Treatment 6 ( Figure 4 ), the proliferation multiple is as high as 20.17 times (Treatment 6 - Y3). The morphological changes during the secondary embryo proliferation in Treatment 6 are as Figure 4 shown, and the proliferation situation of the control group is as Figure 5 shown.

[0107] Without considering the interaction, the factor priority order is 6-BA > NAA > inositol > cyclodextrin. The optimal combination for secondary embryo proliferation is 0.1 mg / L 6-BA + 0.5 mg / L NAA + 600 mg / L inositol + 200 mg / L cyclodextrin.

[0108] Table 4 Effects of hormones and organic additives on the proliferation of secondary embryos

[0109]

[0110] Analysis of variance showed that the hormones 6-BA and NAA had a significant effect on the proliferation of secondary embryos (P<0.05), and the effect of 6-BA on the proliferation of secondary embryos reached an extremely significant level (P<0.01).

[0111] The Duncan method was used to conduct multiple comparisons of the proliferation multiples of secondary embryos under the treatments of 6-BA and NAA (Table 5). As shown in Table 5, low-level (0.1 mg / L) 6-BA significantly promoted the proliferation of secondary embryos, while NAA had the best effect on the proliferation of secondary embryos at the level of 0.5 mg / L.

[0112] Table 5 Duncan multiple comparisons of the proliferation multiples of secondary embryos under the treatments of 6-BA and NAA

[0113]

[0114] Figure 4 The morphological changes during the proliferation of secondary embryos in "Treatment 6" are shown, where:

[0115] a. Day 0 of culture: Initially inoculated secondary embryos.

[0116] b. Day 21 of culture: A large number of secondary embryos proliferated and the volume increased.

[0117] c. Day 30 of culture: The proliferated secondary embryos gradually matured and the volume increased significantly.

[0118] d. Continuing culture for 21 days after removing secondary embryos: After removing some secondary embryos, the remaining embryogenic cells continued to proliferate.

[0119] Figure 5 The morphological changes during the proliferation of secondary embryos in the blank control group (CK) are shown, where:

[0120] a. Day 0 of culture: Initially inoculated secondary embryos.

[0121] b. Day 30 of culture: The secondary embryos in the control group were subjected to conventional proliferation treatment, and there were no obvious changes in volume and quantity.

[0122] Figures 1 to 5 It intuitively shows the indirect and direct pathways of litsea cubeba somatic embryogenesis, as well as the morphological changes and proliferation effects during the proliferation of secondary embryos, providing important visual materials for the research.

[0123] The more suitable subculture period for the proliferation of secondary embryos is about 1 month. The secondary embryos proliferated in 30 days had high activity, and the color of somatic embryos gradually changed from light yellow to white ( Figure 4 in c)), which is a symbol of the gradual maturation and aging of somatic embryos. When the culture time exceeded 30 days, some somatic embryos turned yellowish-brown, and the secondary embryos far from the medium died due to insufficient nutrient supply, while timely removal of mature somatic embryos could promote the continuous proliferation of the remaining embryogenic cells in the dish (Figure 4 in d).

[0124] In the description of the present invention, it should be understood that "-" and "~" represent the range between two numerical values, and this range includes the endpoints. For example: "A-B" represents a range greater than or equal to A and less than or equal to B. "A~B" represents a range greater than or equal to A and less than or equal to B.

[0125] In the description of the present invention, the term "and / or" herein is merely an association relationship describing associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone.

[0126] In the description of the invention, numerical values such as time, temperature, ratio, and mass involved can be based on actual measurements, equipment standard parameters, simplified rounding results, or within an acceptable error range, ensuring the practicability and repeatability of the invention.

[0127] In the description of the present invention, the term "about" or "approximately" is used to express an approximate value of a numerical value or interval, allowing a certain error, to ensure the flexibility and practicability of the description, while remaining within an acceptable error range, and the maximum error range does not exceed 10% of the corresponding numerical value or numerical range.

[0128] The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limitations on the present invention. The protection scope of the present invention should be subject to the scope defined by the claims. For those of ordinary skill in the art, without departing from the spirit and scope of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as within the protection scope of the present invention.

Claims

1. A Litsea cubeba secondary embryo proliferation culture medium, characterized in that, The culture medium formula is as follows: based on the MS medium, add 30 g / L sucrose, 7 g / L agar, 500 mg / L casein hydrolysate, 0.1 mg / L 6-benzylaminopurine, 0.5 mg / L naphthaleneacetic acid, 600 mg / L inositol, 200 mg / L cyclodextrin, and the pH value is 5.6 - 5.

8.

2. The Litsea cubeba secondary embryo proliferation culture medium according to claim 1, wherein The said culture medium is used for the proliferation culture of the secondary embryos of Litsea cubeba.

3. A method for proliferating secondary embryos of Litsea cubeba, characterized in that, It includes the following steps: (a) Somatic embryo formation stage: After disinfecting the explants of immature seeds of Litsea cubeba, inoculate them into the somatic embryo formation medium and conduct dark culture to induce the formation of embryogenic callus and somatic embryos; (b) Secondary embryo proliferation stage: Select somatic embryos at the globular embryo stage and inoculate them into the secondary embryo proliferation medium described in claim 3, and conduct dark culture to complete the proliferation of secondary embryos; (c) Regularly remove mature somatic embryos and conduct subculture once according to a preset period to maintain the continuous proliferation activity of embryogenic cells.

4. The method for proliferating and culturing the secondary embryos of Litsea cubeba according to claim 3, characterized in that, The said somatic embryo formation medium is: based on the MS medium, add 30 g / L sucrose, 7 g / L agar, 700 mg / L casein hydrolysate, 2.0 mg / L 6-benzylaminopurine, 0.6 mg / L 2,4-dichlorophenoxyacetic acid, 0.5 mg / L indoleacetic acid, and the pH value is 5.

8.

5. The method for proliferating and culturing the secondary embryos of Litsea cubeba according to claim 3, characterized in that, In the step (a), the collection time of the said immature seeds is in mid-June, and the disinfection steps include rinsing with running water and soaking in 75% alcohol.

6. The method for proliferating and culturing the secondary embryos of Litsea cubeba according to claim 5, characterized in that, The said disinfection steps are specifically rinsing with running water for 1 h and soaking in 75% alcohol for 3 - 5 min.

7. The method for proliferating and culturing the secondary embryos of Litsea cubeba according to claim 3, characterized in that, In the step (a), conduct dark culture for 90 days, and the incidence rate of cell embryos reaches 23.3%.

8. The method for proliferating and culturing the secondary embryos of Litsea cubeba according to claim 3, characterized in that, In the step (b), the inoculation amount of the said globular embryos is 0.2 - 0.4 g / dish, and the culture temperature is 24 ± 2 °C.

9. The method for proliferating and culturing the secondary embryos of Litsea cubeba according to claim 3, characterized in that, In the step (c), the cycle of the said subculture is 30 days, and calculated by the fresh weight ratio, the proliferation multiple of secondary embryos reaches 20.17 times.

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