A method for inducing mature embryos of Siberian apricot to breed seedlings

Abstract

The invention discloses a method for inducing mature embryos of Siberian apricot to breed seedlings, comprising the following steps: explant disinfection: disinfecting apricot kernels with seed shells removed and peeling off the seed coat to obtain mature embryos; sterile seedling induction: culturing mature embryos in sterile seedling induction medium to obtain sterile seedlings; sterile seedling proliferation: culturing sterile seedlings in sterile seedling proliferation medium to obtain sterile seedlings and strong seedlings; inducing callus: culturing rectangular leaves cut from sterile seedlings and strong seedlings in callus induction medium to obtain callus; differentiating cluster buds: transferring callus to cluster bud differentiation medium to obtain cluster buds; cluster bud subculture proliferation: culturing cluster buds in cluster bud subculture medium after cutting to obtain cluster bud strong seedlings; cluster bud rooting: cutting cluster buds into single buds and transferring them to rooting medium for rooting culture. The invention can solve the technical problems of long breeding cycle, low survival rate and poor breeding stability of Siberian apricot.

Description

Technical Field

[0001] The invention relates to the technical field of Siberian apricot breeding, and more particularly to a method for inducing mature embryos of Siberian apricot to breed seedlings. Background Art

[0002] Siberian apricot (Prunus sibirica) is a shrub or small tree belonging to the genus Prunus of the Rosaceae family. It is an important ecological and economic tree species in my country. Siberian apricot is mainly distributed in the three northern regions of my country, including Inner Mongolia, Liaoning, Hebei, Jilin, Heilongjiang, Gansu and Shanxi.

[0003] Siberian apricot has a well-developed root system and is highly adaptable, cold-resistant, drought-resistant, and barren-resistant. It plays a special role in ensuring the ecological security of northern my country. At the same time, the kernel of Siberian apricot has high nutritional value. The kernel can be processed into almond oil, and the unsaturated fatty acid content in the oil reaches 92%. In addition, its kernel contains trace amounts of amygdalin, which has the effects of relieving cough and reducing phlegm, moisturizing and clearing the lungs. It has a good therapeutic effect on diseases such as asthma and bronchitis. It is also a new type of woody energy plant.

[0004] my country exports about 10,000 tons of almonds each year, but the price of almonds on the market is currently high and in short supply. It is urgent to carry out efficient propagation of Siberian almond varieties and promote their application in production to alleviate the imbalance between supply and demand of almonds. Tissue culture has the characteristics of maintaining the excellent characteristics of tree species and efficient breeding. Therefore, tissue culture and rapid propagation of Siberian apricot germplasm can achieve the purpose of mass production of improved varieties and strong seedlings, which is also a problem that technicians in this field urgently need to solve. Summary of the invention

[0005] To this end, the technical problem to be solved by the present invention is to provide a method for inducing mature embryos of Siberian apricot to breed seedlings, so as to solve the technical problems of long breeding cycle, low survival rate and poor breeding stability of Siberian apricot.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] A method for inducing mature embryos of Siberian apricot to breed seedlings comprises the following steps:

[0008] Step (1), explant disinfection: disinfecting the Siberian apricot kernels after removing the seed coat, and then peeling off the seed coat under sterile conditions to obtain intact mature embryos;

[0009] Step (2), sterile seedling induction: inoculating the mature embryos into a sterile seedling induction medium for light culture to obtain sterile seedlings;

[0010] Step (3), sterile seedling proliferation: cutting the differentiated sterile seedling bud cluster from the base into a number of single sterile seedlings, and then transferring them to a sterile seedling proliferation medium for proliferation culture to obtain sterile seedlings;

[0011] Step (4), inducing callus tissue: cutting rectangular leaves of 1 cm×0.5 cm from sterile seedlings, inoculating the rectangular leaves into callus induction medium for induction culture, and obtaining callus tissue;

[0012] Step (5), differentiating cluster buds: transferring the callus tissue to a cluster bud differentiation medium for differentiation culture to obtain cluster buds;

[0013] Step (6), subculture and proliferation of cluster buds: cutting the differentiated cluster buds into a number of cluster bud individual plants and transferring them to a cluster bud proliferation medium for proliferation culture to obtain cluster bud strong seedlings;

[0014] Step (7), rooting of clustered buds: cutting and separating the clustered buds into single buds, and then transferring the single buds to a rooting medium for rooting culture to obtain Siberian apricot seedlings.

[0015] In the above method for inducing mature embryos of Siberian apricot to breed seedlings, in step (2), the sterile seedling induction medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, 1-naphthylacetic acid (NAA), sucrose and water, and the pH value is 5.75-5.81; each 1L of the sterile seedling induction medium contains 4.3-4.5g of MS medium powder, 5.8-6.0g of agar, 0.5-2.0mg of 6-BA, 0.1-0.2mg of 1-naphthylacetic acid (NAA) and 30g of sucrose. The induction effect of using 6-BA alone on inducing sterile seedlings from mature embryos is significant, while the induction effect of using NAA alone is not significant; with the increase of 6-BA concentration, the differentiation rate of mature embryos increases accordingly, and the number of bud clusters also begins to increase, but when the 6-BA concentration is too high, the bud clusters experience leaf curling, leaf yellowing, or vitrification of leaves to varying degrees in the middle and late stages of culture; the present invention uses 6-BA and NAA in combination and controls the dosage of the two within the above range for inducing sterile seedlings from mature embryos, which can significantly improve the induction rate of inducing sterile seedlings from mature embryos and can effectively avoid adverse phenomena such as leaf curling, yellowing, or vitrification in the middle and late stages of culture.

[0016] The method for inducing mature embryos of Siberian apricot to breed seedlings, in step (3), the sterile seedling proliferation medium is made by mixing MS medium powder, agar, 6-BA, indolebutyric acid (IBA), sucrose and water, and the pH is 5.75-5.81; each 1L of sterile seedling proliferation medium contains 4.3-4.5g of MS medium powder, 5.8-6.0g of agar, 0.8-1.5mg of 6-BA, 0.2-0.5mg of indolebutyric acid and 30g of sucrose. Using 6-BA alone has a significant effect on the proliferation induction of sterile seedlings, and the differentiation rate tends to increase with the increase of its dosage, while the proliferation induction effect of IBA alone is not significant; using 6-BA and IBA together and controlling the dosage of the two within the above range can significantly reduce the dosage of 6-BA and the proliferation rate reaches 100%, and the seedlings grow vigorously and the leaves are dark green. If the 6-BA concentration is less than 0.8 mg / L, even if the IBA dosage is adjusted to the optimal level, the proliferation rate is only 30% to 40%.

[0017] The method for inducing mature embryos of Siberian apricot to breed seedlings, in step (4), the callus induction medium is made by mixing MS medium powder, agar, 6-BA, 2,4-D, sucrose and water, and the pH is 5.75-5.81; each 1L of callus induction medium contains 4.3-4.5g of MS medium powder, 5.8-6.0g of agar, 0.3-1.0mg of 6-BA, 0.5-1.5mg of 2,4-D and 30g of sucrose. The induction rate of leaf callus in lower concentrations of 6-BA and lower concentrations of 2,4-D is low, and the number of calli is small. With the increase of concentration, the induction rate of callus also increases accordingly, but if the concentration is high, it will cause serious browning of leaves and slow growth; the present invention uses 6-BA and 2,4-D in combination, and controls the dosage of the two within the above range, which can significantly increase the induction rate of callus, fast leaf growth, and effectively avoid the appearance of browned leaves.

[0018] In the above method for inducing mature embryos of Siberian apricot to breed seedlings, in step (5), the cluster bud differentiation medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, 2,4-D, sucrose and water, and the pH is 5.75-5.81; each 1L of cluster bud differentiation medium contains 4.3-4.5g of MS medium powder, 5.8-6.0g of agar, 0.5-1.0mg of 6-BA, 0-2.0mg of 2,4-D and 30g of sucrose. In the cluster bud culture medium, an increase in the concentration of 6-BA will lead to serious browning of callus tissue, and the browning rate is the lowest when the 6-BA is 0.5-1.0mg / L; while there is no obvious change when the concentration of 2,4-D is high, so a lower concentration of 6-BA combined with a lower concentration of 2,4-D can significantly reduce the browning of callus tissue and improve the differentiation rate of cluster buds.

[0019] In the method for inducing mature embryos of Siberian apricot to breed seedlings, in step (6), the cluster bud proliferation medium is prepared by uniformly mixing MS medium powder, agar, indolebutyric acid (IBA), 6-BA, sucrose and water, and the pH value is 5.75-5.81; each 1L of cluster bud proliferation medium contains 4.3-4.5g of MS medium powder, 5.8-6.0g of agar, 0.2-0.5mg of indolebutyric acid (IBA), 0.8-1.5mg of 6-BA and 30g of sucrose. With the increase of 6-BA concentration, the cluster bud proliferation rate increases accordingly, and when the 6-BA concentration is 1.5mg / L, the proliferation rate reaches 80%-90%, and when the IBA concentration is higher than 0.5mg / L, the proliferation rate decreases instead.

[0020] In the method for inducing mature embryos of Siberian apricot to breed seedlings, in step (7), the rooting medium is prepared by uniformly mixing 1 / 2MS medium powder, agar, indolebutyric acid (IBA), sucrose and water, and the pH value is 5.75-5.81; each 1L of the rooting medium contains 2.1-2.3g of 1 / 2MS medium powder, 5.8-6.0g of agar, 0.1-1.5mg of indolebutyric acid (IBA) and 30g of sucrose. As the concentration of IBA increases, the rooting rate of the buds increases accordingly. When the concentration of IBA is 1.5mg / L, the rooting rate reaches 80%-90%. When the concentration of IBA is higher than 1.5mg / L, the rooting rate decreases instead, and the average number of roots also decreases.

[0021] In the above method for inducing mature embryos of Siberian apricot to breed seedlings, the culture conditions during the culture in steps (2) to (7) are: light intensity of 2000Lx, light cycle of 16h / d, and light culture temperature of 25±2°C; the culture time of steps (2), (3) and (6) is 15 to 20d; the culture time of step (4) is 20 to 25d; the culture time of step (5) is 35 to 40d; and the culture time of step (7) is 25 to 40d.

[0022] In the above method for inducing mature embryos of Siberian apricot to breed seedlings, in step (1), the method for disinfecting Siberian apricot kernels is as follows: first soak the Siberian apricot kernels in sterile water for 18 to 24 hours, then place them in a clean bench and soak them in 75% alcohol by volume for 30 to 60 seconds, then take them out and rinse them with sterile water for 3 to 5 times, finally soak them in a sodium hypochlorite solution with a mass fraction of 1.5 to 2.0 wt% and shake them for 4 to 8 minutes, then take them out and rinse them with sterile water for 3 to 5 times;

[0023] In step (4), the method for obtaining the rectangular leaves of 1 cm×0.5 cm is as follows: select a strong seedling leaf with good growth, leave a rectangular piece of 1 cm in length in the middle of the leaf, cut off the excess parts on both sides of the leaf to form a rectangle of 1 cm×0.5 cm with wounds on four sides, and use a scalpel to make 3 to 4 wounds on the surface of the leaf; when inoculating, inoculate two rectangular leaves of 1 cm×0.5 cm per bottle, and the sunny side of the rectangular leaves faces upward (the sunny side of the rectangular leaves faces upward to better receive sunlight, promote photosynthesis, and provide more nutrients for the growth of callus tissue; at the same time, it can also increase the temperature of the culture medium, which is conducive to the growth and differentiation of callus tissue; and can reduce the pollution of the culture medium and improve the quality of callus tissue). The present invention cuts the rectangular leaves into leaves of 1 cm×0.5 cm with wounds on four sides and 3 to 4 wounds on the surface, which can increase the contact area between the leaves and the callus induction culture medium, thereby significantly improving the induction efficiency of callus tissue, and at the same time, it is easier to absorb nutrients in the culture medium, which is conducive to the growth and differentiation of callus tissue.

[0024] In the above method for inducing mature embryos of Siberian apricot to breed seedlings, in step (1), the method for disinfecting Siberian apricot kernels is as follows: first soak the Siberian apricot kernels in sterile water for 24 hours, then place them in a clean bench and soak them in 75% alcohol by volume for 30 seconds, then take them out and rinse them with sterile water for 5 times, finally soak them in a sodium hypochlorite solution with a mass fraction of 2.0wt% and shake them for 6 minutes, then take them out and rinse them with sterile water for 5 times;

[0025] In step (2), the sterile seedling induction medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, 1-naphthylacetic acid, sucrose and water, and the pH value is 5.75; each 1L of sterile seedling induction medium contains 4.4g of MS medium powder, 6.0g of agar, 1.0mg of 6-BA, 0.2mg of 1-naphthylacetic acid and 30g of sucrose; the conditions of light culture are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; light culture time is 20d;

[0026] In step (3), the sterile seedling proliferation medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, indolebutyric acid, sucrose and water, and the pH value is 5.78; each 1L of sterile seedling proliferation medium contains 4.4g of MS medium powder, 6.0g of agar, 1.5mg of 6-BA, 0.5mg of indolebutyric acid and 30g of sucrose; the proliferation culture conditions are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; the proliferation culture time is 15d;

[0027] In step (4), the method for obtaining the rectangular leaves of 1 cm×0.5 cm is as follows: select leaves of strong seedlings with good growth, leave a small rectangular piece of 1 cm in the middle of the leaves, cut off the excess parts on both sides of the leaves to form a rectangle of 1 cm×0.5 cm with wounds on four sides, and use a scalpel to make 3 to 4 wounds on the surface of the leaves; when inoculating, inoculate two rectangular leaves of 1 cm×0.5 cm in each bottle, and the sunny side of the rectangular leaves faces upward;

[0028] The callus induction medium is prepared by mixing MS medium powder, agar, 6-BA, 2,4-D, sucrose and water evenly, with a pH of 5.81; each 1L of callus induction medium contains 4.4g of MS medium powder, 6.0g of agar, 0.5mg of 6-BA, 1.0mg of 2,4-D and 30g of sucrose; the induction culture conditions are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2℃; the induction culture time is 25d;

[0029] In step (5), the cluster bud differentiation medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, sucrose and water, and the pH value is 5.80; each 1L of cluster bud differentiation medium contains 4.4g of MS medium powder, 6.0g of agar, 0.5mg of 6-BA and 30g of sucrose; the conditions for differentiation culture are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; the differentiation culture time is 38d;

[0030] In step (6), the cluster bud proliferation medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, indolebutyric acid, sucrose and water, and the pH value is 5.75; each 1L of cluster bud proliferation medium contains 4.4g of MS medium powder, 6.0g of agar, 1.5mg of 6-BA, 0.5mg of indolebutyric acid and 30g of sucrose; the conditions for proliferation culture are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; and proliferation culture time is 15d;

[0031] In step (7), the rooting medium is prepared by uniformly mixing 1 / 2MS medium powder, agar, indolebutyric acid, sucrose and water, and the pH value is 5.76; each 1L of rooting medium contains 2.2g of 1 / 2MS medium powder, 6.0g of agar, 1.0mg of indolebutyric acid and 30g of sucrose; the rooting culture conditions are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; and rooting culture time is 40d.

[0032] The technical solution of the present invention achieves the following beneficial technical effects:

[0033] 1. The disinfection method and culture medium of the present invention are suitable for Siberian apricot, with low contamination rate and high survival rate, so that healthy seedlings can be grown. Moreover, the present invention directly takes the organogenesis pathway as the main method, uses mature embryos to induce sterile seedlings, induces callus through sterile seedling leaves, induces clustered buds through callus, and induces rooting of clustered buds, etc. to obtain in vitro regenerated plants of Siberian apricot, and can obtain a large number of Siberian apricot tissue culture seedlings with strong growth and high survival rate in a short time, which provides sufficient materials for fully exploring the excellent characteristics of Siberian apricot, and at the same time lays a foundation for Siberian apricot variety selection, genetic improvement and quality evaluation.

[0034] 2. The present invention studies the influence of plant nutrient components on the culture effects of different tissue culture stages of Siberian apricot, and comprehensively improves the culture medium components and contents in the sterile seedling induction stage, the sterile seedling proliferation stage, the callus induction stage, the cluster bud differentiation stage, the cluster bud proliferation stage and the cluster bud rooting stage, so that the culture medium in each stage is suitable for the culture medium formula for the differentiation or growth of different tissues of Siberian apricot; the Siberian apricot tissue culture seedlings obtained by culturing with the culture medium of the present invention (sterile seedling induction culture medium, sterile seedling proliferation culture medium, callus induction culture medium, cluster bud differentiation culture medium and cluster bud rooting culture medium) grow robustly and have a high survival rate, providing technical support for the large-scale production of Siberian apricot.

[0035] 3. Compared with the technical scheme of directly using mature embryos to induce callus tissue to redifferentiate and obtain seedlings, the method of the present invention first uses mature embryos to induce sterile seedlings, then uses the leaves of sterile seedlings to induce callus tissue, and finally uses callus tissue differentiation-rooting to obtain Siberian apricot seedlings. The method has the following advantages: (1) Improving induction efficiency: The leaves of sterile seedlings have higher vitality and regeneration ability than mature embryos, and are more likely to form callus tissue. The sterile seedling induction medium, sterile seedling proliferation medium and callus induction medium of the present invention are used to first induce sterile seedlings, and then induce callus tissue from the leaves of sterile seedlings, which can significantly improve the induction efficiency. (1) improving the induction efficiency of callus tissue; (2) improving the adaptability of Siberian seedlings: by first inducing sterile seedlings and then inducing callus tissue from the leaves of the sterile seedlings, the apricot seedlings finally obtained have better adaptability and growth potential than the seedlings obtained by directly using mature embryos to induce callus tissue; this is because the sterile seedlings adapt to the culture environment during the growth process, and the callus tissue induced by their leaves also has better adaptability; (3) reducing risks: there may be some risks in directly using mature embryos to induce callus tissue, such as embryo contamination, genotype restrictions, etc.; and the present invention can reduce these risks and improve the success rate by first inducing sterile seedlings.

[0036] 4. Compared with obtaining sterile seedlings by directly germinating Siberian apricot seeds, the present invention uses mature embryos to obtain sterile seedlings, which has the following advantages: (1) shortening the germination time: mature embryos have gone through a certain development stage, and their germination speed is usually faster than seeds, so the time to obtain sterile seedlings can be significantly shortened; (2) improving the germination rate: the germination rate of mature embryos is usually higher than that of seeds because they already have a certain germination ability; (3) reducing the risk of contamination: mature embryos have undergone certain screening and treatment before germination, and compared with directly using seeds, they carry fewer pathogens and impurities on their surface, thereby reducing the risk of contamination; (4) stable genetic characteristics: mature embryos are separated from the mother plant, and their genetic characteristics are relatively stable, which helps to maintain the genetic consistency of sterile seedlings; (5) facilitating genetic transformation: it is more convenient to use mature embryos for genetic transformation operations because they already have a certain tissue structure and cell differentiation, which can be more easily carried out Gene introduction and cell culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 A real shot of the mature embryos of Siberian apricot cultured in sterile seedling induction medium on the 14th day in Example 2 of the present invention;

[0038] Figure 2 A real shot of the sterile seedlings in Example 2 of the present invention on the 12th day of culture in the sterile seedling proliferation medium;

[0039] Figure 3 A real shot of the leaves of the strong seedlings in Example 2 of the present invention cultured in the callus induction medium on the 21st day;

[0040] Figure 4 A real shot of the callus tissue cultured in the clustered bud differentiation medium on the 35th day in Example 2 of the present invention;

[0041] Figure 5 A real shot of the clustered buds in Example 2 of the present invention on the 13th day of cultivation in the clustered bud proliferation medium;

[0042] Figure 6 A real shot of the clustered shoots in Example 2 of the present invention on the 40th day of culture in the rooting medium. DETAILED DESCRIPTION

[0043] Example 1

[0044] The method of inducing mature embryos of Siberian apricot to breed seedlings in this embodiment comprises the following steps:

[0045] Step (1), explant disinfection: Siberian apricot fruits can be picked after they are ripe, put into a ziplock bag and brought back to the laboratory to manually remove the pulp, and then spread the seeds in a cool and ventilated place to dry. After about a week, they are sealed in a ziplock bag and stored in a refrigerator at 4°C for ready use; the Siberian apricot kernels with the seed shell removed are disinfected, and then the seed coat is peeled off under sterile conditions to obtain intact mature embryos;

[0046] In this embodiment, the method for disinfecting Siberian apricot kernels is as follows: the Siberian apricot kernels are first soaked in sterile water for 24 hours, then placed in a clean bench and soaked in 75% alcohol by volume for 30 seconds, taken out and rinsed with sterile water for 3 times, and finally soaked in a 2.0wt% sodium hypochlorite solution and shaken for 4 minutes, and then taken out and rinsed with sterile water for 3 times.

[0047] Step (2), sterile seedling induction: inoculating intact mature embryos into sterile seedling induction medium for light culture to obtain sterile seedlings;

[0048] In the present embodiment, the sterile seedling induction medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, 1-naphthylacetic acid, sucrose and water, and the pH value is 5.78; each 1L sterile seedling induction medium contains 4.4g of MS medium powder, 6.0g of agar, 0.5mg of 6-BA, 0.1mg of 1-naphthylacetic acid and 30g of sucrose; the conditions of light culture are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2℃; light culture time is 15d.

[0049] Step (3), sterile seedling proliferation: cutting the well-growing and differentiated sterile seedling bud clusters from the base into a number of single sterile seedlings, and then transferring them to a sterile seedling proliferation medium for proliferation culture to obtain sterile seedlings;

[0050] In this embodiment, the sterile seedling proliferation culture medium is prepared by uniformly mixing MS culture medium powder, agar, 6-BA, indolebutyric acid, sucrose and water, and the pH value is 5.77; each 1L of sterile seedling proliferation culture medium contains 4.4g of MS culture medium powder, 6.0g of agar, 0.8mg of 6-BA, 0.2mg of indolebutyric acid and 30g of sucrose; the conditions for proliferation culture are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2℃; and proliferation culture time is 18d.

[0051] Step (4), inducing callus tissue: cutting 1 cm×0.5 cm rectangular leaves with good growth from sterile seedlings, inoculating the rectangular leaves into callus induction medium for induction culture, and obtaining callus tissue;

[0052] In this embodiment, the method for obtaining a 1 cm × 0.5 cm rectangular leaf is as follows: select a strong seedling leaf with good growth, leave a 1 cm long rectangular piece in the middle of the leaf, cut off the excess parts on both sides of the leaf to form a 1 cm × 0.5 cm rectangle with wounds on four sides, and use a scalpel to make 3 to 4 wounds on the surface of the leaf; when inoculating, inoculate two 1 cm × 0.5 cm rectangular leaves per bottle, and the sunny side of the rectangular leaves faces upward;

[0053] The callus induction medium was prepared by mixing MS medium powder, agar, 6-BA, 2,4-D, sucrose and water evenly, with a pH of 5.79; each 1L of callus induction medium contained 4.4g of MS medium powder, 6.0g of agar, 0.3mg of 6-BA, 0.5mg of 2,4-D and 30g of sucrose; the induction culture conditions were: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2℃; the induction culture time was 22d.

[0054] Step (5), differentiating cluster buds: selecting green, compact and soft callus tissue with differentiation ability and transferring it to cluster bud differentiation medium for differentiation culture to obtain cluster buds;

[0055] In this embodiment, the cluster bud differentiation medium is prepared by mixing MS medium powder, agar, 6-BA, 2,4-D, sucrose and water, and the pH is 5.75; each 1L of cluster bud differentiation medium contains 4.4g of MS medium powder, 6.0g of agar, 0.5mg of 6-BA, 0.5mg of 2,4-D and 30g of sucrose; the conditions for differentiation culture are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; differentiation culture time is 35d;

[0056] Step (6), subculture and proliferation of cluster buds: cutting the differentiated cluster buds with good growth into cluster bud individual plants and then transferring them to cluster bud proliferation medium for proliferation culture to obtain cluster bud strong seedlings;

[0057] In this embodiment, the cluster bud proliferation medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, indolebutyric acid, sucrose and water, and the pH value is 5.77; each 1L of cluster bud proliferation medium contains 4.4g of MS medium powder, 6.0g of agar, 0.8mg of 6-BA, 0.2mg of indolebutyric acid and 30g of sucrose; the conditions for proliferation culture are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2℃; and proliferation culture time is 18d.

[0058] Step (7), rooting of clustered buds: select clustered bud seedlings with good differentiation morphological characteristics (strong clustered buds, green leaves, about 5 cm long), cut and separate them into single buds, and then transfer the single buds to a rooting medium for rooting culture to obtain Siberian apricot seedlings.

[0059] In this embodiment, the rooting medium is prepared by uniformly mixing 1 / 2MS medium powder, agar, indolebutyric acid, sucrose and water, and the pH value is 5.80; each 1L of rooting medium contains 2.2g of 1 / 2MS medium powder, 6.0g of agar, 0.1mg of indolebutyric acid and 30g of sucrose; the rooting culture conditions are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; the rooting culture time is 35d.

[0060] Example 2

[0061] The method for inducing mature embryos of Siberian apricot to breed seedlings in this embodiment is different from that in embodiment 1 in that: in step (1), the method for disinfecting Siberian apricot kernels is as follows: the Siberian apricot kernels are first soaked in sterile water for 24 hours, then placed in a clean bench and soaked in 75% alcohol by volume for 30 seconds, then taken out and rinsed with sterile water for 5 times, finally soaked in a sodium hypochlorite solution with a mass fraction of 2.0wt% and shaken for 6 minutes, then taken out and rinsed with sterile water for 5 times;

[0062] In step (2), the sterile seedling induction medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, 1-naphthylacetic acid, sucrose and water, and the pH value is 5.75; each 1L of sterile seedling induction medium contains 4.4g of MS medium powder, 6.0g of agar, 1.0mg of 6-BA, 0.2mg of 1-naphthylacetic acid and 30g of sucrose; the conditions of light culture are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; light culture time is 20d;

[0063] In step (3), the sterile seedling proliferation medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, indolebutyric acid, sucrose and water, and the pH value is 5.78; each 1L of sterile seedling proliferation medium contains 4.4g of MS medium powder, 6.0g of agar, 1.5mg of 6-BA, 0.5mg of indolebutyric acid and 30g of sucrose; the proliferation culture conditions are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; the proliferation culture time is 15d;

[0064] In step (4), the callus induction medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, 2,4-D, sucrose and water, and the pH value is 5.81; each 1L of callus induction medium contains 4.4g of MS medium powder, 6.0g of agar, 0.5mg of 6-BA, 1.0mg of 2,4-D and 30g of sucrose; the induction culture conditions are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; the induction culture time is 25d;

[0065] In step (5), the cluster bud differentiation medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, sucrose and water, and the pH value is 5.80; each 1L of cluster bud differentiation medium contains 4.4g of MS medium powder, 6.0g of agar, 0.5mg of 6-BA and 30g of sucrose; the conditions for differentiation culture are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; the differentiation culture time is 38d;

[0066] In step (6), the cluster bud proliferation medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, indolebutyric acid, sucrose and water, and the pH value is 5.77; each 1L of cluster bud proliferation medium contains 4.4g of MS medium powder, 6.0g of agar, 0.8mg of 6-BA, 0.2mg of indolebutyric acid and 30g of sucrose; the conditions for proliferation culture are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2℃; and proliferation culture time is 18d.

[0067] In step (7), the rooting medium is prepared by uniformly mixing 1 / 2MS medium powder, agar, indolebutyric acid, sucrose and water, and the pH value is 5.76; each 1L of rooting medium contains 2.2g of 1 / 2MS medium powder, 6.0g of agar, 1.0mg of indolebutyric acid and 30g of sucrose; the rooting culture conditions are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; and rooting culture time is 40d.

[0068] Example 3

[0069] The method for inducing mature embryos of Siberian apricot to breed seedlings in this embodiment is different from that in embodiment 1 in that: in step (1), the method for disinfecting Siberian apricot kernels is as follows: the Siberian apricot kernels are first soaked in sterile water for 24 hours, then placed in a clean bench and soaked in 75% alcohol by volume for 60 seconds, then taken out and rinsed with sterile water for 3 times, finally soaked in a sodium hypochlorite solution with a mass fraction of 2.0wt% and shaken for 8 minutes, then taken out and rinsed with sterile water for 5 times;

[0070] In step (2), the seedling induction medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, 1-naphthylacetic acid, sucrose and water, and the pH value is 5.76; each 1L of sterile seedling induction medium contains 4.4g of MS medium powder, 6.0g of agar, 2.0mg of 6-BA, 0.2mg of 1-naphthylacetic acid and 30g of sucrose; the conditions of light culture are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; light culture time is 18d;

[0071] In step (3), the sterile seedling proliferation medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, indolebutyric acid, sucrose and water, and the pH value is 5.78; each 1L of sterile seedling proliferation medium contains 4.4g of MS medium powder, 6.0g of agar, 1.5mg of 6-BA, 0.5mg of indolebutyric acid and 30g of sucrose; the proliferation culture conditions are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; the proliferation culture time is 18d;

[0072] In step (4), the callus induction medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, 2,4-D, sucrose and water, and the pH value is 5.75; each 1L of callus induction medium contains 4.4g of MS medium powder, 6.0g of agar, 1.0mg of 6-BA, 1.5mg of 2,4-D and 30g of sucrose; the induction culture conditions are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; the induction culture time is 22d;

[0073] In step (5), the cluster bud differentiation medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, sucrose and water, and the pH value is 5.78; each 1L of cluster bud differentiation medium contains 4.4g of MS medium powder, 6.0g of agar, 1.0mg of 6-BA, 2.0mg of 2,4-D and 30g of sucrose; the conditions for differentiation culture are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; the differentiation culture time is 40d;

[0074] In step (6), the cluster bud proliferation medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, indolebutyric acid, sucrose and water, and the pH value is 5.77; each 1L of cluster bud proliferation medium contains 4.4g of MS medium powder, 6.0g of agar, 0.8mg of 6-BA, 0.2mg of indolebutyric acid and 30g of sucrose; the conditions for proliferation culture are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2℃; and proliferation culture time is 18d.

[0075] In step (7), the rooting medium is prepared by uniformly mixing 1 / 2MS medium powder, agar, indolebutyric acid, sucrose and water, and the pH value is 5.75; each 1L of rooting medium contains 2.2g of 1 / 2MS medium powder, 6.0g of agar, 1.5mg of indolebutyric acid and 30g of sucrose; the rooting culture conditions are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; and the rooting culture time is 42d.

[0076] Comparative Example

[0077] The difference between this comparative example and Example 1 is that the sterile seedling induction culture in step (2), the sterile seedling proliferation medium in step (3), the callus induction medium in step (4), the cluster bud differentiation medium in step (5) and the rooting medium in step (7) are all commonly used ordinary MS medium (pH is controlled in the range of 5.75-5.81, and each 1L of MS medium contains 5.8-6.0g of agar and 4.4g of MS medium powder). The remaining operating methods and operating conditions are the same as those in Example 1.

[0078] Compared with Example 1, in this comparative example, at the sterile seedling induction stage: the contamination rate of mature embryos on the ordinary MS culture medium was high, the differentiation rate was low, the number of bud clusters was small, and the differentiation rate was 10%; at the sterile seedling proliferation stage: the yellowing phenomenon of the sterile seedlings was common, the growth was slow, and the proliferation effect was poor; at the leaf callus induction stage: there was no obvious differentiation reaction and the survival rate was low; at the cluster bud differentiation stage: the cluster buds were not differentiated after 40 days of cultivation, and the browning phenomenon was serious; at the cluster bud rooting stage: the rooting rate was only 10%, and the root system was in poor condition.

[0079] Example 1 In the sterile seedling induction stage: the sterile seedlings grew vigorously, the leaves were dark green, the growth was good, the induction rate was as high as 82%, and the contamination rate was low (11%); the sterile seedling proliferation stage: the proliferation rate was 43%, most of them differentiated into cluster buds, and only a few differentiated into axillary buds; the leaf callus induction stage: the differentiation rate was 61%, the differentiation started from the leaf edge, gradually spread to the inside, and finally the leaf completely became callus; the cluster bud differentiation stage: the seedlings were differentiated after 35 days of cultivation, and the differentiation rate was high (76%); the cluster bud rooting stage, the rooting rate could reach up to 84%, and the root growth required about 60 days.

[0080] Example 2 In the sterile seedling induction stage: the sterile seedlings grew vigorously, the leaves were dark green, the growth was good and the induction rate was as high as 80%, and the contamination rate was low (5%); the sterile seedling proliferation stage: the proliferation rate was 100%, most of them differentiated into cluster buds, and only a few differentiated into axillary buds; the leaf callus induction stage: the differentiation rate was 70%, the differentiation started from the leaf edge, gradually spread to the inside, and finally the leaf completely became callus; the cluster bud differentiation stage: the seedlings were differentiated after 35 days of cultivation, and the differentiation rate was high (85%); the cluster bud rooting stage, the rooting rate could reach up to 90%, but the root system grew slowly and needed about 35 days.

[0081] Compared with Example 1 and Example 2, in Example 3, during the sterile seedling induction stage: the disinfection time was longer, resulting in a lower induction rate (35%); during the sterile seedling proliferation stage: the proliferation rate was lower (33%) and the seedling height was lower; during the leaf callus induction stage: the callus was severely browned and grew slowly, with a differentiation rate of 26%; during the cluster bud differentiation stage: the callus structure was relatively loose, and no cluster buds were differentiated after 40 days of inoculation; during the cluster bud rooting stage, no rooting was observed after 40 days of culture.

[0082] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A method for inducing mature embryos of Siberian apricot to breed seedlings, characterized in that: The steps include: Step (1), explant disinfection: disinfecting the Siberian apricot kernels after removing the seed coat, and then peeling off the seed coat under sterile conditions to obtain intact mature embryos; Step (2), sterile seedling induction: inoculating mature embryos into sterile seedling induction medium for light culture to obtain sterile seedlings; the sterile seedling induction medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, 1-naphthylacetic acid, sucrose and water, and the pH value is 5.75-5.81; each 1L of the sterile seedling induction medium contains 4.3-4.5g of MS medium powder, 5.8-6.0g of agar, 0.5-2.0mg of 6-BA, 0.1-0.2mg of 1-naphthylacetic acid and 30g of sucrose; Step (3), sterile seedling proliferation: cutting the differentiated sterile seedling buds from the base into individual sterile seedlings, and then transferring them to a sterile seedling proliferation medium for proliferation culture to obtain sterile seedlings; the sterile seedling proliferation medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, indolebutyric acid, sucrose and water, and the pH value is 5.75 to 5.81; each 1L of the sterile seedling proliferation medium contains 4.3 to 4.5g of MS medium powder, 5.8 to 6.0g of agar, 0.8 to 1.5mg of 6-BA, 0.2 to 0.5mg of indolebutyric acid and 30g of sucrose; Step (4), inducing callus tissue: cutting rectangular leaves of 1 cm×0.5 cm from sterile seedlings, inoculating the rectangular leaves into callus tissue induction medium for induction culture, and obtaining callus tissue; the callus tissue induction medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, 2,4-D, sucrose and water, and the pH value is 5.75-5.81; each 1L of callus tissue induction medium contains 4.3-4.5g of MS medium powder, 5.8-6.0g of agar, 0.3-0.5mg of 6-BA, 0.5-1.0mg of 2,4-D and 30g of sucrose; Step (5), differentiating cluster buds: transferring the callus tissue to a cluster bud differentiation medium for differentiation culture to obtain cluster buds; the cluster bud differentiation medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, 2,4-D, sucrose and water, and the pH value is 5.75-5.81; each 1L of the cluster bud differentiation medium contains 4.3-4.5g of MS medium powder, 5.8-6.0g of agar, 0.5-1.0mg of 6-BA, 0-0.5mg of 2,4-D and 30g of sucrose; Step (6), subculture and proliferation of cluster buds: cutting the differentiated cluster buds into cluster bud individual plants and transferring them to a cluster bud proliferation medium for proliferation culture to obtain cluster bud seedlings; the cluster bud proliferation medium is prepared by uniformly mixing MS culture medium powder, agar, indolebutyric acid, 6-BA, sucrose and water, and the pH value is 5.75-5.81; each 1L of the cluster bud proliferation medium contains 4.3-4.5g of MS culture medium powder, 5.8-6.0g of agar, 0.2-0.5mg of indolebutyric acid, 0.8-1.5mg of 6-BA and 30g of sucrose; Step (7), rooting of clustered buds: cutting and separating the clustered bud seedlings into single buds, and then transferring the single buds to a rooting medium for rooting culture, thereby obtaining Siberian apricot seedlings; the rooting medium is prepared by uniformly mixing 1 / 2MS medium powder, agar, indolebutyric acid, sucrose and water, and the pH value is 5.75-5.81; each 1L of the rooting medium contains 2.1-2.3g of 1 / 2MS medium powder, 5.8-6.0g of agar, 0.1-1.5mg of indolebutyric acid and 30g of sucrose.

2. The method for inducing mature embryos of Siberian apricot to breed seedlings according to claim 1, characterized in that: The culture conditions during the culture in steps (2) to (7) are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; the culture time of steps (2), (3) and (6) is 15 to 20 days; the culture time of step (4) is 20 to 25 days; the culture time of step (5) is 35 to 40 days; and the culture time of step (7) is 25 to 40 days.

3. The method for inducing mature embryos of Siberian apricot to breed seedlings according to claim 1, characterized in that: In step (1), the method for disinfecting Siberian apricot kernels is as follows: first soak the Siberian apricot kernels in sterile water for 18 to 24 hours, then place them in a clean bench and soak them in 75% alcohol by volume for 30 to 60 seconds, then take them out and rinse them with sterile water for 3 to 5 times, finally soak them in a sodium hypochlorite solution with a mass fraction of 1.5 to 2.0 wt% and shake them for 4 to 8 minutes, then take them out and rinse them with sterile water for 3 to 5 times; In step (4), the method for obtaining the 1 cm×0.5 cm rectangular leaves is as follows: select leaves of strong seedlings with good growth, leave a 1 cm long rectangular piece in the middle of the leaves, cut off the excess parts on both sides of the leaves to form a 1 cm×0.5 cm rectangle with wounds on all four sides, and use a scalpel to make 3 to 4 wounds on the surface of the leaves; when inoculating, inoculate two 1 cm×0.5 cm rectangular leaves in each bottle, and the sunny side of the rectangular leaves faces upward.

4. The method for inducing mature embryos of Siberian apricot to breed seedlings according to any one of claims 1 to 3, characterized in that: In step (1), the method for disinfecting Siberian apricot kernels is as follows: first soak the Siberian apricot kernels in sterile water for 24 hours, then place them in a clean bench and soak them in 75% alcohol by volume for 30 seconds, then take them out and rinse them with sterile water for 5 times, then soak them in a 2.0wt% sodium hypochlorite solution and shake them for 6 minutes, then take them out and rinse them with sterile water for 5 times; In step (2), the sterile seedling induction medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, 1-naphthylacetic acid, sucrose and water, and the pH value is 5.75; each 1L of sterile seedling induction medium contains 4.4g of MS medium powder, 6.0g of agar, 1.0mg of 6-BA, 0.2mg of 1-naphthylacetic acid and 30g of sucrose; the sterile seedling induction culture conditions are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; light culture time of 20d; In step (3), the sterile seedling proliferation medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, indolebutyric acid, sucrose and water, and the pH value is 5.78; each 1L of sterile seedling proliferation medium contains 4.4g of MS medium powder, 6.0g of agar, 1.5mg of 6-BA, 0.5mg of indolebutyric acid and 30g of sucrose; the conditions for the sterile seedling proliferation culture are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; and proliferation culture time of 15d; In step (4), the method for obtaining the 1 cm × 0.5 cm rectangular leaves is as follows: select leaves of strong seedlings with good growth, leave a 1 cm long rectangular piece in the middle of the leaves, cut off the excess parts on both sides of the leaves to form a 1 cm × 0.5 cm rectangle with wounds on four sides, and use a scalpel to make 3 to 4 wounds on the surface of the leaves; when inoculating, inoculate two 1 cm × 0.5 cm rectangular leaves in each bottle, and the sunny side of the rectangular leaves faces upward; The callus induction medium is prepared by mixing MS medium powder, agar, 6-BA, 2,4-D, sucrose and water evenly, with a pH of 5.81; each 1L of callus induction medium contains 4.4g of MS medium powder, 6.0g of agar, 0.5mg of 6-BA, 1.0mg of 2,4-D and 30g of sucrose; the conditions for callus induction culture are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2℃; induction culture time of 25d; In step (5), the cluster bud differentiation medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, sucrose and water, and the pH value is 5.80; each 1L of cluster bud differentiation medium contains 4.4g of MS medium powder, 6.0g of agar, 0.5mg of 6-BA and 30g of sucrose; the conditions for cluster bud differentiation culture are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; differentiation culture time is 38d; In step (6), the cluster bud proliferation medium is prepared by uniformly mixing MS medium powder, agar, 6-BA, indolebutyric acid, sucrose and water, and the pH value is 5.75; each 1L of cluster bud proliferation medium contains 4.4g of MS medium powder, 6.0g of agar, 1.5mg of 6-BA, 0.5mg of indolebutyric acid and 30g of sucrose; the conditions for cluster bud proliferation culture are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; proliferation culture time is 15d; In step (7), the rooting medium is prepared by uniformly mixing 1 / 2MS medium powder, agar, indolebutyric acid, sucrose and water, and the pH value is 5.76; each 1L of the rooting medium contains 2.2g of 1 / 2MS medium powder, 6.0g of agar, 1.0mg of indolebutyric acid and 30g of sucrose; the rooting culture conditions are: light intensity of 2000Lx, light cycle of 16h / d, light culture temperature of 25±2°C; and rooting culture time of 40d.

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