Methods for improving the induction rate of embryonic callus of Pinus elliottii or Pinus elliottii

Abstract

The present application relates to the field of forest tree seedling breeding biotechnology, and specifically to a method for improving the induction rate of embryonic callus of Pinus elliottii or Pinus elliottii, comprising picking cones that meet preset conditions from a donor plant, separating seeds from the cones, peeling off female gametophytes from the seeds, and inducing and culturing the female gametophytes using somatic embryogenesis technology; the preset conditions include: (1) the donor plant is in the induction harvest period; (2) the development status of the picked cones is qualified; the present application solves the problems of poor repeatability and failure of somatic embryogenesis caused by misunderstandings in the traditional method in the judgment of embryonic callus induction and the calculation of induction rate, and provides an important reference for the large-scale production of somatic embryo seedlings of Pinus elliottii or Pinus elliottii based on somatic embryogenesis technology.

Description

Technical Field

[0001] The present application relates to the field of forest tree seedling propagation biotechnology, and in particular to a method for improving the induction rate of embryonic callus of Pinus elliottii or Pinus elliottii. Background Art

[0002] Longleaf pine (Pinus elliottii Engelm.) is a large evergreen tree of the genus Pinus in the Pinaceae family. It has a straight trunk and can be used for construction, sleepers, pit wood, and raw materials for wood fiber and papermaking industries. Longleaf pine (Pinus elliottii × P. caribaea) is a hybrid of longleaf pine and Caribbean pine, also known as hybrid pine or hybrid pine. Its trunk is round and straight, and its basic wood density and fiber length are between the two parents. It is resistant to water and humidity, grows rapidly, has strong adaptability, strong wind resistance, and strong resistance to pests and diseases. It has a wide adaptability and has extremely high economic value.

[0003] The main way to achieve asexual reproduction of Pinus elliottii or Pinus elliottii through biotechnology is somatic embryogenesis. To achieve this technology, it is necessary to first induce embryonic callus. The induction rate of embryonic callus is affected by many factors, among which genotype and the development stage of zygotic embryos are the two most critical factors. It is known that different genotypes and different reciprocal cross combinations have a great influence on the induction rate. These factors can be improved by screening highly inductive genotypes and reciprocal cross combinations, but in the actual application of somatic embryogenesis, only highly inductive genotypes cannot be used to produce somatic embryos. The zygotic embryo development stage is one of the important factors that can be judged and controlled by some means. At present, many studies believe that the second to fourth stages of the zygotic embryo development stage have a better induction effect, but most studies only stay at the comparison of fruit picking time, and obtain the result that the cones harvested on a certain date have a better induction rate. Usually, this result has very limited guiding significance. Because of the changes in annual temperature and precipitation, the cone development speed varies by about 1-2 weeks each year, and the window period for the best induction rate is also 1-2 weeks. Therefore, the time node of the best induction rate in a certain year is not necessarily the best induction time node for the next year. Moreover, even for mother trees of the same asexual line, due to differences in their geographical location in the seed garden, differences in the pollen genotypes they receive, and the attachment positions and orientations of different cones on the tree body, they all affect the development stage of the zygotic embryo, resulting in different cones having different zygotic embryo development stages, making the induction rate show mother tree and cone-dependent differences, that is, seeds from different mother trees and different cones will have large differences in induction rates.

[0004] Currently, there is no universal method for inducing embryonic callus. Moreover, based on the above reasons, the currently known methods for obtaining embryonic callus from Pinus elliottii or Pinus elliottii are very likely to have errors in the identification of embryonic callus induction and the calculation of induction rate. This leads to poor reproducibility of the results when these methods are implemented, which also has a great impact on the realization of the subsequent somatic embryogenesis process. Summary of the invention

[0005] In order to solve the above technical problems, one or more embodiments of the present application provide a method for improving the induction rate of embryonic callus of Pinus elliottii or Pinus elliottii.

[0006] The method for improving the induction rate of embryonic callus of Pinus elliottii or Pinus elliottii comprises picking cones that meet preset conditions from a donor plant, separating seeds of the cones, peeling off female gametophytes of the seeds, and inducing and culturing the female gametophytes using somatic embryogenesis technology;

[0007] The preset conditions include:

[0008] (1) The donor plant is in the induction harvesting period;

[0009] (2) The development status of the picked cones is satisfactory;

[0010] The method for determining the induced harvest period is as follows:

[0011] Pick cones from different plants 2 to 3 weeks before the induction harvest period, separate seeds from the cones, peel off the female gametophytes of the seeds, longitudinally cut the female gametophytes, and divide the seeds into A1 seeds, B1 seeds, C1 seeds, and D1 seeds according to the cross-sectional morphology of the longitudinal sections, and calculate the percentage of each type of seeds;

[0012] If half or more of the collected cones have the following characteristics (1) to (3), they have reached the induced harvest period. Otherwise, they are observed once a week until they reach the induced harvest period:

[0013] (1) The percentage of A1 seeds is 0% to 15%,

[0014] (2) The sum of the percentages of B1 seeds and C1 seeds is 50% to 100%,

[0015] (3) The percentage of C1 seeds contained is 25% to 100%;

[0016] The A1 seeds have at least one of the following characteristics: 1) no groove in the middle of the cross section, 2) the immature zygotic embryo is small and easy to break and cannot be separated, 3) there is no obvious visible embryo head; the B1 seeds have at least three of the following characteristics: 1) the groove in the middle of the cross section is visible, 2) the immature zygotic embryo can be separated, 3) the embryo head is transparent and visible, 4) the suspensor is transparent and visible and connected to the embryo head; the C1 seeds have at least three of the following characteristics: 1) the groove in the middle of the cross section is clearly visible, 2) the immature zygotic embryo can be separated, 3) the embryo head is clearly visible and the shape is basically the same as the attached Figure 2 The "bullet head" shape shown in stage III is white and opaque. 4) The embryo suspensor is clearly visible and connected to the embryo head. The shape is basically the same as in the attached figure. Figure 2 The lower part of the "bullet head" shown in stage III is translucent; the D1 seeds have at least one of the following characteristics: 1) the female gametophyte is wrinkled, 2) the female gametophyte is abnormally gray-brown in color, 3) does not contain a female gametophyte, 4) the groove in the middle of the cross section is clearly visible but there is no zygotic embryo;

[0017] The formula for calculating the percentage of each type of seed is as follows:

[0018] The percentage of A1 seeds = [the number of A1 seeds ÷ (the total number - the number of D1 seeds)] × 100%,

[0019] The percentage of B1 seeds = [the number of B1 seeds ÷ (the total number - the number of D1 seeds)] × 100%,

[0020] The percentage of C1 seeds = [number of C1 seeds ÷ (total number - number of D1 seeds)] × 100%,

[0021] The method for judging whether the developmental status is qualified is as follows:

[0022] Pick cones from different plants in the induction harvest period, separate seeds from the cones, peel off the female gametophytes of the seeds, longitudinally cut the female gametophytes, and divide the seeds into A2 seeds, B2 seeds, C2 seeds, and D2 seeds according to the cross-sectional morphology of the longitudinal sections, and calculate the percentage of each type of seeds;

[0023] The classification standards and percentage calculation formulas of the A2 seeds, B2 seeds, C2 seeds and D2 seeds are the same as those of the A1 seeds, B1 seeds, C1 seeds and D1 seeds respectively;

[0024] The seeds in the cones collected for observation have the following characteristics (4) to (6), that is, the development status is qualified, otherwise they are discarded:

[0025] (4) The percentage of A2 seeds contained is 0%~15%,

[0026] (5) The sum of the percentages of B2 and C2 seeds is 40% to 100%.

[0027] (6) The percentage of C2 seeds contained is 20%~100%. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 This is a schematic diagram of the process of screening mother trees and single cones and inducing embryonic callus tissue of Pinus elliottii and Pinus elliottii in one embodiment of the present application;

[0030] Figure 2 Schematic diagram of various developmental stages of the zygotic embryo of a seed in one embodiment of the present application;

[0031] Figure 3 This is a schematic diagram of a method for efficiently stripping female gametophytes from sterile seeds in one embodiment of the present application;

[0032] Figure numerals: 1 is a gun-shaped tweezers; 2 is a sterilized seed; x, y, z respectively indicate three different force application directions. DETAILED DESCRIPTION

[0033] The present application will be further described below in conjunction with the embodiments and examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. In addition, it should be understood that after reading the content taught in the present application, those skilled in the art may make various changes or modifications to the present application, and these equivalent forms also fall within the protection scope of the claims attached to the present application.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0035] the term:

[0036] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0037] In this application, the term "about" is generally used in conjunction with a numerical value and can refer to a set or range of values. For example, "about X" includes a range of values ​​of ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of X, where X is a numerical value.

[0038] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0039] Herein, "preferred", "preferable", "better", etc. are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0040] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0041] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0042] In this application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0043] The components of the mDCR basic culture medium in the present application can be referred to Table 1, which can be homemade according to the medium formula, or directly purchased from the market.

[0044] Table 1 Composition of mDCR basic medium

[0045]

[0046] In one aspect of the present application, a method for inducing embryonic callus of Pinus elliottii and Pinus elliottii is provided.

[0047] The technical personnel of the present application conducted a large amount of literature research during the cultivation of the embryonic callus tissue of Pinus elliottii and Pinus elliottii, and unexpectedly found that the cultivation according to the "high induction rate" technical scheme recorded in the prior art could not produce mature plants with high yields. After research, it was believed that there might be misunderstandings in the identification of the embryonic callus tissue of Pinus elliottii and Pinus elliottii in the prior art, which led to the fact that the method could not actually produce the embryonic callus tissue of Pinus elliottii and Pinus elliottii without any doubt, which led to the subsequent breeding failure.

[0048] The embryonic callus tissue obtained by traditional methods is generally "false", or a mixture of "false" and "true", that is, true embryonic and non-true embryonic cultures are mixed together. The probability of embryonic loss in the subsequent proliferation process is greater. In many cases, these induced products cannot continue to proceed and complete the somatic embryogenesis process. Even if these products are mistakenly further matured and cultured, it will only lead to the failure of maturation culture and the failure to obtain mature somatic embryos.

[0049] Longleaf pine and Longleaf pine belong to the genus Pinus. Their embryonic callus induction usually starts from the female gametophyte. The induction step sometimes only induces the immature zygotic embryo mass to eject from the micropyle, but does not produce embryonic callus that can proliferate spontaneously. The criterion for distinguishing these two tissue masses is whether they can continue to proliferate over time, and the embryonic callus usually comes from the ejected immature zygotic embryo mass. The actual induction rate of embryonic callus should be based on ejection and proliferation. However, in some technologies, the tissue of non-zygotic embryos in the female gametophyte is induced by hormones to produce non-embryonic callus, which sometimes grows into relatively large masses. In this case, non-embryonic callus is induced at the non-micropyle ejection position, and the induction rate is calculated based on this, but the subsequent culture cannot successfully and efficiently proliferate or obtain somatic embryos. If the cell morphology is observed microscopically, it is found that there is no embryo head and suspensor structure. Non-real embryonic callus cannot obtain mature somatic embryos, which makes the previous work completely in vain and wastes a lot of manpower and material costs. Therefore, how to obtain "true embryonic callus" (that is, embryonic callus that can be used for further culture into mature somatic embryos) is the key to increasing the yield of somatic embryos and somatic embryo seedlings.

[0050] Therefore, the criteria for judging the generation of embryonic callus in this application are as follows: ① ejection from the micropylar end of the megagametophyte; ② continued proliferation over time after ejection; ③ presence of embryo head and suspensor structure observed by microscopic staining. In this application, the induced products that meet the above criteria are selected to calculate the true embryonic callus induction rate, and the calculated induction rate is improved compared to the traditional technology, and the success rate of culturing mature somatic embryos based on the obtained embryonic callus is also improved.

[0051] The technical solution of the present application can effectively screen out cone seeds that are not suitable for induction, reduce the damage caused by stripping female gametophytes on the clean bench, improve the work efficiency of stripping female gametophytes for induction, adjust and optimize the culture medium formula to improve the universality of the induction culture medium, thereby improving the induction rate of true embryonic callus. In addition, the present application also provides a discrimination system for the zygotic embryo development stage suitable for embryonic callus induction of slash pine and wetland pine, as well as a method for identifying embryonic callus and calculating the true induction rate, providing a certain basis for the development of related research.

[0052] The technical solution of this application is basically based on the growth characteristics of embryonic callus. Figure 1 Please refer to the flowchart below for further explanation.

[0053] An embodiment of the present application provides a method for inducing embryonic callus tissue of Pinus elliottii or Pinus elliottii, which comprises the following steps: determining an asexual mother tree for induction, then judging whether the mother tree has reached the induction harvesting time, then harvesting fruits in large quantities at the optimal harvesting time, then judging the developmental stage of a single cone, then disinfecting seeds, then aseptically and efficiently extracting female gametophytes without damage, then performing callus induction, then performing callus proliferation and embryonic staining judgment, and finally calculating the embryonic callus induction rate.

[0054] It is important to note that determining whether a mother tree has reached the time for induced harvesting is a continuous process, and there are the following methods and standards for determination:

[0055] Zygotic embryo development stage

[0056] The traditional zygotic embryo development stage discrimination system divides the developmental stages of zygotic embryos into 7-9 or more stages, but in fact only the earlier zygotic embryos have induction potential. It is necessary to reset a zygotic embryo development stage discrimination system for embryonic callus induction.

[0057] Based on this, the present application divides the early zygotic embryo development into 4 stages, plus the empty grain classification, a total of 4 stages and 5 classifications, and uses an ordinary stereo microscope for observation.

[0058] Stage I (seeds at this stage are also referred to as A1 seeds or A2 seeds in this article. A1 and A2 are used to distinguish seeds from different sources, but both are at stage I and have the same characteristics). Seeds at this stage have at least one of the following characteristics: 1) no groove in the middle of the cross section, 2) the immature zygotic embryo is small and easy to break and cannot be separated, 3) there is no obvious embryo head; further, seeds at this stage have the following characteristics: when the female gametophyte is cut longitudinally in half, there is no groove in the middle or the groove is very shallow, the immature zygotic embryo cannot be separated or after separation, it appears as very thin hair-like, and there is no obvious embryo head;

[0059] Stage II (seeds at this stage are also referred to as B1 seeds or B2 seeds in this article. B1 and B2 are used to distinguish seeds from different sources, but both are at stage II and have the same characteristics). Seeds at this stage have at least three of the following characteristics: 1) a groove is visible in the middle of the cross section, 2) an immature zygotic embryo can be separated, 3) the embryo head is transparent and visible, 4) the suspensor is transparent and visible and connected to the embryo head; further, seeds at this stage have the following characteristics: when the female gametophyte is cut longitudinally in half, there is a shallow groove in the middle, and the immature zygotic embryo can be separated, with a very small embryo head at the top, and the suspensor is visible and connected to the embryo head, both of which are almost transparent;

[0060] Stage III (seeds at this stage are also referred to as C1 seeds or C2 seeds in this article. C1 and C2 are used to distinguish seeds from different sources, but both are at stage III and have the same characteristics). Seeds at this stage have at least three of the following characteristics: 1) the groove in the middle of the cross section is clearly visible, 2) the immature zygotic embryo can be separated, 3) the embryo head is clearly visible and the morphology is basically the same as the attached Figure 2 The "bullet head" shape shown in stage III is white and opaque. 4) The embryo suspensor is clearly visible and connected to the embryo head. The shape is basically the same as in the attached figure. Figure 2 The lower part of the "bullet head" shown in stage III is shaped and translucent; further, the lower part of the "bullet head" refers to the slightly swollen position next to the lower part of the bullet head structure; further, the seeds at this stage have the following characteristics: the female gametophyte is cut longitudinally in half, the middle groove is obvious, and the immature zygotic embryo can be separated, and the top has a "bullet head" shaped embryo head, which is longer than that in stage II, the embryo suspensor is clearly visible and connected to the embryo head, the top of the embryo head is relatively transparent, the lower part is slightly whitish, and the embryo suspensor is transparent or translucent;

[0061] Stage IV: The isolated immature zygotic embryos have an obviously whitish and longer embryo head, the top of the embryo head begins to flatten or the cotyledon structure begins to form, and the suspensor begins to shrink and degenerate. All stages from the top of the embryo head flattening to the cotyledons fully developed and the suspensor completely degenerated are classified as stage IV;

[0062] "Empty grains" classification (seeds with this classification feature are also referred to as D1 seeds or D2 seeds in this article. D1 and D2 are used to distinguish seeds from different sources, but both have the same classification features of this category); during the observation process, if the female gametophyte is shriveled, small and not full, abnormal color, etc., or the empty seeds are found to contain no female gametophyte after opening, they should be classified as empty grains; the seeds at this stage have at least one of the following features: 1) the female gametophyte is shriveled, 2) the female gametophyte is abnormally gray-brown in color, 3) does not contain a female gametophyte, 4) the groove in the middle of the cross section is clearly visible but there is no zygotic embryo. See the schematic diagram of each stage for details. Figure 2 .

[0063] Prediction of the best induced harvesting time for mother trees

[0064] According to the growth characteristics of pine plants, the best harvesting time of mother trees can be reasonably predicted to improve the efficiency of induction work. The collection of immature cones and the prediction of the development of this year for slash pine and wetland pine usually start in mid-to-early June each year. For different asexual mother trees, the harvesting time can be determined based on the situation in previous years, and the collection and prediction are usually carried out 2-3 weeks before the best induction harvesting time in previous years.

[0065] The collection and prediction methods can be: ① According to the needs of this year, select fixed mother trees for fruit collection, which can be open-pollinated mother trees or controlled-pollinated mother trees. The prediction results of each mother tree cannot be referenced with each other; ② Select 3-5 immature cones located at the top of the mother tree and facing south. After harvesting, peel off the female gametophyte to determine the zygotic embryo development stage. Each cone should have no less than 50 seeds (if the entire cone has less than 50 seeds, all should be observed). The zygotic embryo development stage identification system is used for identification, and the zygotic embryo development stage of each seed is recorded. ③ Count and calculate the percentage of each development stage of each cone according to the following formula: percentage of A1 seeds = [number of A1 seeds ÷ (total number - number of D1 seeds)] × 100%, percentage of B1 seeds = [number of B1 seeds ÷ (total number - number of D1 seeds)] × 100%, percentage of C1 seeds = [number of C1 seeds ÷ (total number - number of D1 seeds)] × 100%; ④ Check the distribution of seed quantity at each stage of the observed cones. For Pinus elliottii, in the determination of the induced harvest period, if half or more of the cones collected and observed have the following characteristics (7) to (9), the induced harvest period has been reached. Otherwise, observation is conducted once a week until the induced harvest period is reached: (7) the percentage of A1 seeds contained is 0% to 15%, (8) the sum of the percentage of B1 seeds and C1 seeds contained is 60% to 100%, (9) the percentage of C1 seeds contained is 30% to 100%. For Pinus elliottii, in the determination of the induced harvest period, if half or more of the cones collected and observed have the following characteristics (13) to (1 5) characteristics, that is, reaching the induced harvest period, otherwise observe once a week until reaching the induced harvest period: (13) the percentage of A1 seeds contained is 0~15%, (14) the sum of the percentage of B1 seeds and C1 seeds contained is 50%~100%, (15) the percentage of C1 seeds contained is 25%~100%; ⑤ For mother trees that have not reached the optimal harvest time, the fruit should be harvested and observed once every week, using the same method as before; ⑥ When it is judged that the mother trees have reached the optimal harvest time, batch harvesting is carried out at one time, and the collected immature cones are placed in a 4-degree refrigerator and stored in a sealed bag.

[0066] After harvesting fruits in large quantities at the optimal harvesting time, it is still necessary to identify the development stage of individual cones. This is because even if the zygotic embryo development stage of the mother trees is predicted, some of the cones harvested in batches will still develop too fast or too slowly and should be discarded after identification to increase the induction rate.

[0067] Single cone development stage identification and selection criteria

[0068] The cones were opened with the assistance of patented devices (ZL 202021011527.1) and patented devices (ZL 202021011890.3), and the seeds were manually peeled out. Depending on the number of seeds in a single cone, about 20% of the seeds were randomly selected, and the female gametophyte was peeled to determine the zygotic embryo development stage. The determination and calculation of the developmental stage of a single cone is described in "Prediction of the optimal induction and harvesting time of mother trees". Specifically, the developmental stage standard of the single cone selected for induction is: for slash pine, the seeds in the cones collected for observation have the following characteristics (4) to (6), that is, the developmental status is qualified, otherwise it is discarded: (4) The percentage of A2 seeds contained is 0 ~15%, (5) the sum of the percentage of B2 seeds and C2 seeds contained is 50%~100%, (6) the percentage of C2 seeds contained is 25%~100%; the seeds in the cones collected and observed for wet pine have the following characteristics (16)~(18), that is, the development state is qualified, otherwise they are discarded: (16) the percentage of A2 seeds contained is 0~15%, (17) the sum of the percentage of B2 seeds and C2 seeds contained is 40%~100%, (18) the percentage of C2 seeds contained is 20%~100%. The remaining seeds of the selected single cones can be stripped of wings and directly disinfected and induced, or stripped of wings and stored in sterile water at 4℃ overnight for induction. The seeds of the unselected cones are discarded.

[0069] Seed disinfection, sterile and efficient non-destructive stripping of female gametophytes

[0070] Take the selected single cone seeds, rinse them with sterile water on a clean bench, disinfect them with 10% 84 disinfectant (containing 0.2% Tween-20) for 10 minutes, rinse them several times, disinfect them with 20% hydrogen peroxide for 10 minutes, rinse them clean and set them aside; because it is difficult to peel the sterile female gametophyte on the bench and it is easy to damage the endosperm, a method for efficiently and non-destructively peeling the sterile female gametophyte on the clean bench was developed (see the schematic diagram Figure 3 ), the key points include: ① All items that come into contact with the sterilized seeds should be sterilized by high-pressure steam or high-temperature sterilization, including pad paper, tweezers, scalpels, etc.; ② The seeds are placed horizontally and upright, with the pointed side facing up and the arc side facing down, and clamped to the position with the anti-slip strips on the front end of the 20cm gun-shaped tweezers; Use a scalpel to adjust the position of the seeds at the front end of the tweezers to the position as shown in the attached picture. Figure 3 ③ Clamp the tweezers to the middle turning position of the gun-shaped tweezers and close them until they can no longer be closed. Use a scalpel to split the obtuse triangle vertically from the middle and move it to the left and right. ④ The seed coat and the testa are separated to both sides, exposing the female gametophyte for peeling. Furthermore, after peeling out the complete female gametophyte quickly and non-destructively, place it carefully and gently on the surface of the induction medium for induction.

[0071] Wound healing induction

[0072] The induction rate of embryonic callus is affected by the components of the induction medium, including mineral elements, organic acids, vitamins, carbon source sugars, plant hormones, various additives, etc., especially the types and levels of hormones (or described as plant growth regulators) in the medium. Different types and concentration combinations of auxins and cytokinins have a significant effect on the speed and induction rate of induction initiation. This effect has been reported in a large number of studies. Choosing the right combination and concentration of auxins and cytokinins can increase the induction rate, but this increase has an upper limit. After obtaining the relatively optimal combination, the induction rate cannot be further increased; abscisic acid plays a key role in both zygotic embryo development and somatic embryo development, but this role has different effects in different species, and other plant hormones and additives need to be considered comprehensively so that the entire formula can achieve the best induction effect.

[0073] An embodiment of the present application provides an induction culture medium, which includes an mDCR basic culture medium, a nitrogen source, a carbon source, activated carbon and a growth regulator; the growth regulator includes abscisic acid, naphthylacetic acid, 6-benzylaminopurine, 6-furfurylaminopurine, abscisic acid and 24-epibrassinolide. The induction culture medium has a good induction effect on the female gametophytes of Pinus elliottii and Pinus elliottii, with less loss and high induction rate.

[0074] Activated carbon is rarely used in induction culture medium due to its broad-spectrum and efficient adsorption of hormones (or growth regulators). The appropriate amount of activated carbon used in one embodiment of the present application was unexpectedly found to produce good results in the induction cultivation of Pinus elliottii and Pinus elliottii.

[0075] The induction medium of one embodiment of the present application includes 5 mg / L to 7.5 mg / L of abscisic acid and 0.1 g / L to 0.2 g / L of activated carbon;

[0076] Further, it also includes 1.5 mg / L to 3 mg / L of naphthylacetic acid, 0.5 mg / L to 1 mg / L of 6-benzylaminopurine, 0.5 mg / L to 1 mg / L of 6-furfurylaminopurine and 0.48 mg / L to 0.96 mg / L of 24-epicrassinolide;

[0077] Furthermore, it also includes 0.3g / L~0.5g / L of hydrolyzed casein, 15g / L~20g / L of maltose, 0mg / L~0.1mg / L of xylose and 450mg / L~650mg / L of L-glutamine.

[0078] Further, the induction medium was composed as follows: mDCR basic medium containing 0.3 g / L~0.5 g / L hydrolyzed casein, 15 g / L~20 g / L maltose, 0 mg / L~0.1 mg / L xylose, 1.5 mg / L~3 mg / L naphthylacetic acid, 0.5 mg / L~1 mg / L 6-benzylaminopurine, 0.5 mg / L~1 mg / L 6-furfurylaminopurine, 5 mg / L~7.5 mg / L abscisic acid, 0.48 mg / L~0.96 mg / L 24-epibrassinolide, 0.1 g / L~0.25 g / L 2-(N-morpholino)ethanesulfonic acid, 450 mg / L~650 mg / L L-glutamine, 0.1 g / L~0.2 g / L activated carbon and 2.5 g / L~3 g / L phytagel.

[0079] Callus proliferation and embryogenic staining identification, statistics of embryogenic callus induction rate

[0080] After about 20 days of induction culture, translucent ejecta can be observed at the micropyle. After 1-1.5 months of continuous culture, some ejecta can be observed to begin to proliferate. The proliferating ejecta are transferred to the proliferation medium, one seed per mass, one to one. When they grow into a mass the size of a fingernail, they are stained with 2% acetic acid magenta and placed under a microscope for identification. The mass with an embryo head formed by dense cells and an embryo stalk formed by long strip cells, and the two connected, is the embryonic callus tissue successfully induced. The number of such masses is recorded, and the true embryonic callus induction rate is calculated.

[0081] The true embryonic callus induction rate of one embodiment of the present application = (the number of embryonic callus clumps ÷ the number of seeds inoculated for induction) × 100%.

[0082] Further explanation of the somatic embryo culture method for the clones of Pinus elliottii

[0083] In one embodiment of the present application, the slash pine clones A04 and A05 have three mother trees in different locations of the seed garden, which are intended to be used for embryonic callus induction, codenamed A04-1, A04-2, A04-3 and A05-1, A05-2, A05-3. When the embryonic callus induction work was carried out in the first year, the technical solution of the present application was not used. From June 10 to June 30, four fruit pickings were carried out, with 3-5 fruits picked each time, and then induced uniformly. The female gametophyte was severely browned, and a small amount of light yellow mass was obtained. After continued cultivation, the embryonic callus was stained and identified, and the induction rate was calculated. The overall induction rate of A04-1 was 2.22% (that is, 23 masses of embryonic callus were obtained by inducing 1036 seeds), and the induction rate of A04-2 was 4. The induction rate of A04-3 was 3.56% (i.e., 43 clusters of embryonic callus were obtained from 1207 seeds), the induction rate of A05-1 was 1.78% (i.e., 21 clusters of embryonic callus were obtained from 1180 seeds), the induction rate of A05-2 was 1.56% (i.e., 13 clusters of embryonic callus were obtained from 833 seeds), and the induction rate of A05-3 was 0.88% (i.e., 10 clusters of embryonic callus were obtained from 1134 seeds). However, if the induction rates of different fruit picking times, different mother trees and different cones were counted separately, the highest could reach 22.4% and the lowest was 0%. At the same time, many clusters could not continue to proliferate and died during subsequent subculture.

[0084] When we continued this work the following year, we used the method of predicting the best harvesting time for different mother trees and found that A05-3 developed the slowest, and its best harvesting time was around July 13th of that year, while A05-1 and A05-2 developed at similar speeds, and their best harvesting time was around July 5th of that year. The best harvesting time for A04-1 and A04-3 was around June 28th of that year, and the best harvesting time for A04-2 was around June 22nd of that year. We collected cones in large quantities at the best harvesting time of each mother tree, and identified and screened the developmental stages of the harvested individual cones. We counted the induction of the batch of cones harvested from each mother tree at its best harvesting time, separated the embryonic callus of single seeds one by one, and calculated the embryonic callus induction rate based on the embryonic callus clumps that could proliferate normally and were identified by staining. After adopting the above methods, the induction rate of cones harvested at the optimal harvesting time was significantly improved. Overall, the induction rate of A04-1 reached 17.04%, the induction rate of A04-2 reached 16.67%, the induction rate of A04-3 reached 14.08%, the induction rate of A05-1 reached 19.33%, the induction rate of A05-2 reached 18.48%, and the induction rate of A05-3 reached 15.66%, and the embryonic callus masses proliferated well.

[0085] When this work was carried out in the third year, the culture medium formula and the method of aseptic stripping of female gametes were further improved.

[0086] In one embodiment of the present application, some cones were selected from multiple mother trees to test the effects of various basic culture media including the original basic culture medium, mLV basic culture medium, and mDCR basic culture medium on the induction rate, among which the original basic culture medium had an average induction rate of 9.04% in multiple asexual mother trees, the mLV basic culture medium had an average induction rate of 4.54% in multiple asexual mother trees, and the mDCR basic culture medium had an average induction rate of 21.55% in multiple asexual mother trees. The induction effect of the mDCR basic culture medium was better.

[0087] In one embodiment of the present application, some cones were selected from multiple mother trees to test the effect of adding ABA in the culture medium on induction. When no ABA was added to the culture medium, the average induction rate of the multiple mother trees was 4.08%. After adding 5 mg / L ABA, the average induction rate of the multiple mother trees was 12.58%. After adding 7.5 mg / L ABA, the average induction rate of the multiple mother trees was 16.45%. Adding 5-7.5 mg / L ABA can increase the induction rate by 3-4 times, but further increasing the ABA concentration in the culture medium has no significant effect on the induction rate.

[0088] In one embodiment of the present application, some cones were selected from multiple mother trees to test the effect of adding activated carbon to the culture medium on induction. When no activated carbon was added to the culture medium, the average induction rate of multiple mother trees was 13.01%, but the occurrence of non-embryonic callus and browning was also relatively high. When 0.1 g / L of activated carbon was added to the culture medium, the average induction rate of multiple mother trees was 17.86%, and the occurrence of non-embryonic callus and browning was greatly reduced, about half. When 0.2 g / L of activated carbon was added to the culture medium, the average induction rate of multiple mother trees was 15.63%, and the occurrence of non-embryonic callus and browning was greatly reduced. The occurrence of embryonic callus and browning was also reduced by about half. When 0.4g / L of activated carbon was added to the culture medium, the average induction rate of multiple mother trees was 4.25%. At this time, the induction rate had begun to be affected. When 0.8g / L of activated carbon was added to the culture medium, the average induction rate of multiple mother trees was 1.15%, and the induction rate was significantly reduced. Adding a small amount of 0.1-0.2g / L of activated carbon to the culture medium can greatly reduce the incidence of non-embryonic callus and browning, and at the same time increase the induction rate. However, continuing to increase the amount of activated carbon added will affect the induction rate and reduce it.

[0089] In one embodiment of the present application, a method for efficiently and non-destructively stripping sterile female gametophytes in an ultra-clean workbench is also developed, and training is conducted among multiple experimental personnel. Generally, the method can be mastered after 2-3 exercises after training. The efficiency of stripping female gametophytes can be increased from 20-30 seeds per person per hour, with a success rate of only 60-70% (some seeds will pop out and be lost outside the filter paper, and some seeds will be accidentally cut), to 90-100 seeds per person per hour, with a success rate of more than 80%. The use of this stripping method further reduces the induction rate of non-embryonic callus tissue. The induction rate of non-embryonic callus tissue in the control group is 88.13%, and the induction rate of non-embryonic callus tissue in the new stripping method is 71.90%.

[0090] Further explanation of the somatic embryo culture method for the clones of Pinus solani

[0091] In one embodiment of the present application, there are multiple mother trees in the seed garden for the asexual lines of Pinus solani G5-3, G5-4 and A05×H06 that can be used for embryonic callus induction. In the first year, one mother tree was selected from the seed garden for each of the three asexual lines for embryonic callus induction. Since the optimal harvesting time of the mother trees was not determined, the induction rate of embryonic callus was very low. The overall average induction rate of G5-4 was only 1.46%, and the overall average induction rate of A05×H06 was only 1.14%. No embryonic callus was obtained from G5-3.

[0092] The following year, two mother trees were selected from each of the three clones for embryonic callus induction, and the codes were G5-3①, G5-3②, G5-4①, G5-4②, A05×H06-1, and A05×H06-2. The best harvest time for all three mother trees was determined, and it was found that the best harvest time for G5-3① was around June 13, the best harvest time for G5-3② was around June 19, and the best harvest time for G5-4① and G5-4② was around June 19. Around June 29, the best harvesting time for A05×H06-1 and A05×H06-2 that year was around July 5. Cones were collected in large quantities at the best harvesting time for each mother tree, and the developmental stages of the harvested individual cones were identified and screened. The induction status of the cones harvested from each mother tree at its best harvesting time was counted, and the embryonic callus of single seeds was separated one by one. The embryonic callus induction rate was calculated based on the embryonic callus masses that could proliferate normally and were identified by staining. After adopting the above method, the induction rate of cones harvested at the optimal harvesting time was significantly improved. Overall, the induction rate of G5-3① reached 13.02%, the induction rate of G5-3② reached 8.89%, the induction rate of G5-4① reached 7.77%, the induction rate of G5-4② reached 21.47%, the induction rate of A05×H06-1 reached 7.39%, and the induction rate of A05×H06-2 reached 13.33%. Although some embryonic callus clumps turned brown and died, the number of proliferation was much higher than that in the first year.

[0093] In the third year, as with the longleaf pine, we selected some cones from different mother trees at the best harvesting stage to optimize the culture medium formula and aseptic stripping methods.

[0094] In one embodiment of the present application, the most suitable basic culture medium for wet and loose is also mDCR, with an average induction rate of 9.05% (the average induction rate of the original basic culture medium is 6.67%, and the average induction rate of mLV basic culture medium is 0.23%).

[0095] In one embodiment of the present application, the addition of ABA can also significantly increase the average induction rate of wet-added pine embryonic callus, from 0.75% when no ABA is added to 4.58% (5 mg / L ABA) and 8.87% (7.5 mg / L ABA). When added to 7.5 mg / L ABA, the increase in the induction rate is more obvious.

[0096] In one embodiment of the present application, the addition of activated carbon does not significantly improve the induction rate of wet callus. When no activated carbon is added, the average induction rate is 14.52%, when 0.1 g / L activated carbon is added, the average induction rate is 15.76%, and when 0.2 g / L activated carbon is added, the average induction rate is 13.11%. When activated carbon is continued to be added, the average induction rate is significantly reduced to 4.24% (0.4 g / L activated carbon) and 0.97% (0.7 g / L activated carbon). However, since the addition of activated carbon can still significantly reduce the induction rate of non-embryonic callus and the incidence of browning, it is recommended to add a small amount of activated carbon, not exceeding 0.1 g / L.

[0097] In one embodiment of the present application, the method of efficiently and non-destructively stripping sterile female gametophytes in an ultra-clean workbench is also applicable to wet-added pine, which can significantly improve the efficiency of stripping female gametophytes, reduce the damage rate of endosperm, and reduce the induction rate of non-embryonic callus to a certain extent.

[0098] In one embodiment of the present application, two mother trees of the slash pine clones A01 and A06 are respectively provided at different locations in the seed garden for embryonic callus induction, codenamed A01-1 and A01-2, A06-1 and A06-2. The present application was used when the embryonic callus induction work was carried out in the first year, wherein the best harvesting time of the A01-1 mother tree in that year was around July 3, the best harvesting time of the A01-2 mother tree in that year was around July 10, and the best harvesting time of the A06-1 and A06-2 mother trees in that year was around July 15. Cones were collected in large quantities at the best harvesting time of each mother tree, and the developmental stages of the harvested individual cones were identified and screened, the induction conditions of the batch of cones harvested from each mother tree at its best harvesting time were counted, the embryonic callus of single seeds were separated one by one, and the embryonic callus induction rate was calculated based on the embryonic callus masses that can proliferate normally and are identified by staining. Overall, the induction rates of A01-1, A01-2, A06-1, A06-2, and A06-1 reached 14.56%, 17.78%, 24.64%, and 15.22%, respectively, and a considerable number of embryonic clones were successfully obtained.

[0099] In one embodiment of the present application, there are multiple mother trees in the seed garden for the wet pine asexual lines A05×Q17, A05×H3, G2-1, G3-1 and G4-1 that can be used for embryonic callus induction. When the embryonic callus induction work was carried out in the first year, the present application was used. For each of the five asexual lines, one mother tree was selected from the seed garden, and the optimal harvesting time of the mother tree was first determined. The best harvesting time of the mother tree selected by A05×Q17 was around July 5, the best harvesting time of the mother tree selected by A05×H3 was around July 10, the best harvesting time of the mother tree selected by G2-1 was around June 28, the best harvesting time of the mother tree selected by G3-1 was around June 23, and the best harvesting time of the mother tree selected by G4-1 was around June 30. Cones were collected in large quantities at the best harvesting time of each mother tree, and the developmental stages of the harvested individual cones were identified and screened. The induction conditions of the batch of cones harvested from each mother tree at its best harvesting time were counted, and single-seed embryonic callus were separated one by one. The induction rate of embryonic callus was calculated based on the embryonic callus masses that could proliferate normally and were identified by staining. Overall, the induction rates of A05×Q17 reached 22.86%, A05×H3 reached 17.36%, G2-1 reached 12.29%, G3-1 reached 10.99%, and G4-1 reached 12.67%, and a considerable number of embryonic asexual lines were successfully obtained.

[0100] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0101] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0102] The above-described embodiments only express several implementation methods of the present application, but they cannot be understood as limiting the scope of the patent application. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present application, several variations and improvements can also be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solutions provided in the present application through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for increasing the induction rate of embryonic callus of Pinus elliottii or Pinus elliottii, characterized in that: The method comprises picking cones from donor plants in the induction harvesting period, selecting cones that meet preset conditions, separating seeds of the cones, peeling off female gametophytes of the seeds, and inducing and culturing the female gametophytes by somatic embryogenesis technology; The preset conditions include: The cones are in good condition; The method for determining the induced harvest period is as follows: Referring to the best induction harvesting time in previous years, cones of different plants were picked 2 to 3 weeks before the best induction harvesting time, seeds of the cones were separated, female gametophytes of the seeds were peeled off, the female gametophytes were longitudinally cut, and the seeds were divided into A1 seeds, B1 seeds, C1 seeds and D1 seeds according to the cross-sectional morphology of the longitudinal section, and the percentage of each type of seeds was calculated; The donor plant is Pinus elliottii. If more than half of the cones collected and observed have the following characteristics (7) to (9), it has reached the induced harvest period. Otherwise, it is observed once a week until it reaches the induced harvest period: (7) The percentage of A1 seeds contained is 0%~15%, (8) The sum of the percentages of B1 and C1 seeds is 60% to 100%. (9) The percentage of C1 seeds contained is 30% to 100%; The donor plant is a wet-added pine. If more than half of the cones collected and observed have the following characteristics (13) to (15), they have reached the induced harvest period. Otherwise, they are observed once a week until they reach the induced harvest period: (13) The percentage of A1 seeds contained is 0%~15%, (14) The sum of the percentages of B1 and C1 seeds is 50% to 100%. (15) The percentage of C1 seeds contained is 25%~100%; The A1 seeds have at least one of the following characteristics: 1) no groove in the middle of the cross section, 2) the immature zygotic embryo is small and easy to break and cannot be separated, 3) there is no obvious visible embryo head; The B1 seeds have at least three of the following characteristics: 1) the groove in the middle of the cross section is visible, 2) the immature zygotic embryo can be separated, 3) the embryo head is transparent and visible, 4) the embryo suspensor is transparent and visible and connected to the embryo head; The C1 seeds have at least three of the following characteristics: 1) the groove in the middle of the cross section is clearly visible, 2) the immature zygotic embryo can be separated, 3) the embryo head is clearly visible and has a "bullet head" shape, is white and opaque, and 4) the embryo suspensor is clearly visible and connected to the embryo head, has a "bullet head" shape at the bottom, and is translucent; The D1 seeds have at least one of the following characteristics: 1) the female gametophyte is wrinkled, 2) the female gametophyte is abnormally gray-brown in color, 3) does not contain a female gametophyte, 4) the groove in the middle of the cross section is clearly visible but there is no zygotic embryo; The formula for calculating the percentage of each type of seed is as follows: The percentage of A1 seeds = [the number of A1 seeds ÷ (the total number - the number of D1 seeds)] × 100%, The percentage of B1 seeds = [the number of B1 seeds ÷ (the total number - the number of D1 seeds)] × 100%, The percentage of C1 seeds = [number of C1 seeds ÷ (total number - number of D1 seeds)] × 100%, The method for judging whether the cone development state is qualified is as follows: Pick cones from different plants in the induction harvest period, separate 20% of the seeds from the cones, peel off the female gametophytes of the seeds, longitudinally cut the female gametophytes, and divide the seeds into A2 seeds, B2 seeds, C2 seeds, and D2 seeds according to the cross-sectional morphology of the longitudinal sections, and calculate the percentage of each type of seeds; The classification standards and percentage calculation formulas of the A2 seeds, B2 seeds, C2 seeds and D2 seeds are the same as those of the A1 seeds, B1 seeds, C1 seeds and D1 seeds respectively; The donor plant is Pinus elliottii. The seeds in the cones collected for observation have the following characteristics (10) to (12), that is, the cones are judged to be in a qualified development state and the remaining seeds in the cones are used for subsequent induction culture, otherwise they are discarded: (10) The percentage of A2 seeds contained is 0%~15%, (11) The sum of the percentages of B2 seeds and C2 seeds is between 50% and 100%, and, (12) The percentage of C2 seeds contained is 25% to 100%; The donor plant is Pinus solani, and the seeds in the cones collected for observation have the following characteristics (16) to (18), that is, the cone development status is judged to be qualified and the remaining seeds in the cones are used for subsequent induction culture, otherwise they are discarded: (16) The percentage of A2 seeds contained is 0%~15%, (17) The sum of the percentages of B2 seeds and C2 seeds is between 40% and 100%, and, (18) The percentage of C2 seeds contained is 20%~100%.

2. The method for improving the induction rate of embryonic callus of Pinus elliottii or Pinus elliottii as claimed in claim 1, characterized in that: The method of separating the seeds of the cones and stripping the female gametophytes of the seeds comprises: Lay the peeled seeds horizontally and upright with the pointed side facing upwards and the arc side facing downwards, and clamp them to the position with anti-slip strips on the front end of the 20 cm gun-shaped tweezers; use a scalpel to adjust the position of the seeds at the front end of the tweezers, and adjust the state. At this time, most of the seeds are hidden in the tweezers, and only the tip of the pointed side is exposed, forming an obtuse triangle; clamp the tweezers to the middle turning position of the gun-shaped tweezers and close them until they can no longer be closed, use a scalpel to split the obtuse triangle vertically in the middle and move it left and right; separate the seed shell and the outer seed coat to both sides, exposing the female gametophyte, and peel them.

3. The method for improving the induction rate of embryonic callus of Pinus elliottii or Pinus elliottii as claimed in claim 2, characterized in that: Before stripping the female gametophyte of the seeds, the seeds are first disinfected, including: Rinse once with sterile water, then soak in 84 disinfectant with a volume concentration of 8%~10% and 0.2% Tween-20 for 9min~11min, then rinse with sterile water 3~4 times, then soak in 20%~25% hydrogen peroxide solution for disinfection by shaking for 9min~11min, then rinse with sterile water 4-5 times until there is no obvious foam.

4. The method for improving the induction rate of embryonic callus of Pinus elliottii or Pinus elliottii as described in any one of claims 1 to 3, characterized in that: The induction culture of the female gametophyte using somatic embryogenesis technology comprises: Cultivating the female gametophyte using an induction medium; The induction medium comprises mDCR basic medium, a nitrogen source, a carbon source, activated carbon and a growth regulator; The growth regulators include abscisic acid, naphthylacetic acid, 6-benzylaminopurine, 6-furfurylaminopurine, abscisic acid and 2,4-epibrassinolide.

5. The method for improving the induction rate of embryonic callus of Pinus elliottii or Pinus elliottii as claimed in claim 4, characterized in that: The nitrogen source is hydrolyzed casein and L-glutamine; and / or The carbon source is maltose and xylose; and / or The pH of the induction medium is 5.7-5.

8.

6. The method for improving the induction rate of embryonic callus of Pinus elliottii or Pinus elliottii as claimed in claim 4, characterized in that: The induction medium satisfies at least one of the following conditions: (1) Including 5 mg / L~7.5 mg / L abscisic acid and 0.1 g / L~0.2 g / L activated carbon; (2) Including 1.5mg / L~3mg / L of naphthylacetic acid, 0.5mg / L~1mg / L of 6-benzylaminopurine, 0.5mg / L~1mg / L of 6-furfurylaminopurine and 0.48mg / L~0.96mg / L of 2,4-epibrassinolide; (3) Includes 0.3 g / L~0.5 g / L of hydrolyzed casein, 15 g / L~20 g / L of maltose, 0 mg / L~0.1 mg / L of xylose and 450 mg / L~650 mg / L of L-glutamine.

7. The method for improving the induction rate of embryonic callus of Pinus elliottii or Pinus elliottii as claimed in claim 6, characterized in that: The composition of the induction medium is as follows: mDCR basic medium and added with 0.3g / L~0.5g / L hydrolyzed casein, 15g / L~20g / L maltose, 0mg / L~0.1mg / L xylose, 1.5mg / L~3mg / L naphthaleneacetic acid, 0.5mg / L~1mg / L 6-benzylaminopurine, 0.5mg / L~1mg / L 6-furfurylaminopurine, 5mg / L~7.5mg / L abscisic acid, 0.48mg / L~0.96mg / L 2,4-epibrassinolide, 0.1g / L~0.25g / L 2-(N-morpholino)ethanesulfonic acid, 450mg / L~650mg / L L-glutamine, 0.1g / L~0.2g / L activated carbon and 2.5g / L~3g / L phytagel.

8. The method for increasing the induction rate of embryonic callus of Pinus elliottii or Pinus elliottii as claimed in any one of claims 1 to 3 and 5 to 7, characterized in that: After inducing and culturing the female gametophyte using somatic embryogenesis technology, identifying whether the induced product belongs to embryonic callus tissue includes: The product obtained by the induction is taken, dyed with a dye, and then transferred to a microscope for observation. If an embryo head formed by dense cells and an embryo suspensor formed by long cells are observed and the two are connected, it is identified that the obtained product belongs to embryonic callus.

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