Culture medium and method for obtaining apricot and plum hybrid seedlings by embryo rescue

Abstract

The invention provides a culture medium and a method for obtaining prunus armeniaca hybrid seedlings by embryo rescue, and belongs to the technical field of plant breeding. The invention provides a culture medium combination for obtaining prunus armeniaca hybrid seedlings by using an embryo rescue method, a culture medium contains main culture mediums required in a period from embryo germination to adventitious bud differentiation, and contents of the culture mediums are optimized. The invention further provides an embryo rescue method matched with the culture medium combination for use, the embryo rescue method can effectively promote continuous growth of apricot and plum hybrid embryos under the in-vitro condition, the germination rate of the embryos is increased, raw materials are provided for obtaining more hybrid progenies, the embryo rescue seedling base number of the apricot and plum hybrid progenies is greatly increased, the method operability is high, and the method is suitable for large-scale popularization and application. The mastering is easy.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and in particular relates to a culture medium and a method for obtaining apricot-plum hybrid seedlings by utilizing embryo rescue. Background Art

[0002] The new hybrid variety of apricot plum (Prunus domestica × armeniaca) is an emerging fruit obtained through multi-generation hybrid breeding with plum and apricot as parents in the United States. The fruit has a unique strong aroma and a higher sugar content than apricot or plum varieties, and is a fruit with good market prospects. However, conventional hybrid breeding has a long cycle and has different degrees of embryo abortion. Therefore, rescuing hybrid embryos before hybrid embryos abort is an important way to obtain distant hybrid apricot plum seedlings.

[0003] In most cases, there is a certain degree of reproductive isolation between distantly related species and genera, which may lead to the failure of the young embryo to develop, abnormal development or stop midway after fertilization; or the lack of coordination between the young embryo, endosperm and ovary tissues of hybrid seeds, especially abnormal endosperm development, which affects the normal development of the embryo. Therefore, it is necessary to study the breeding of new hybrid varieties of apricot and plum in order to obtain distant hybrids. Summary of the invention

[0004] The invention provides a culture medium and a method for obtaining apricot-plum hybrid seedlings by using embryo rescue, which effectively promotes the germination of young embryos and the breeding of seedlings and improves the germination rate of young embryos rescued by embryos.

[0005] The present invention provides a culture medium combination for obtaining apricot-plum hybrid seedlings by using an embryo rescue method, comprising an embryo germination culture medium, a callus induction culture medium and an adventitious bud differentiation culture medium;

[0006] The embryo germination medium uses MS medium as the basic medium and also includes 1-2 mg / L 6-BA, 0.5-1 mg / L IAA and 20-50 g / L sucrose;

[0007] The callus induction medium uses MS medium as the basic medium and also includes 2 mg / L 6-BA and 0.5 mg / L NAA;

[0008] The adventitious bud differentiation medium uses MS medium as the basic medium and further comprises 0.5 mg / L NAA and 0.5-2.0 mg / L 6-BA.

[0009] In a specific embodiment of the present invention, MS culture medium is also included.

[0010] The present invention also provides a method for obtaining embryo rescue of apricot-plum hybrid seedlings, comprising the following steps: (1) after cross-pollination of apricot-plum varieties, picking fruits at least 60 days after pollination, removing immature embryos after disinfection, and inoculating the immature embryos onto an embryo germination medium in the above-mentioned culture medium combination to induce germination of the immature embryos;

[0011] (2) The germinated embryos are inoculated onto MS medium and cultured for a period of time, and then transferred to callus induction medium to induce callus;

[0012] (3) The callus tissue is inoculated onto an adventitious bud differentiation medium to obtain test tube plantlets.

[0013] In a specific embodiment of the present invention, the disinfection in step (1) comprises washing the seeds taken from the fruit with sterile water, and then soaking them in a 70% by volume alcohol aqueous solution, rinsing them with sterile water, soaking them in mercuric chloride, and rinsing them with sterile water.

[0014] In a specific embodiment of the present invention, the temperature for inducing the germination of the young embryos in step (1) is 25±2°C for 25 to 30 days; the illumination time is 16h / d, and the illumination intensity is 2000 to 3000lx.

[0015] In a specific embodiment of the present invention, step (2) is carried out by dark culturing on MS culture medium, and the dark culturing time is 30 days.

[0016] In a specific embodiment of the present invention, the callus induction in step (2) is carried out in dark culture at a temperature of 25±2° C. for 30 to 40 days.

[0017] In a specific embodiment of the present invention, the culture temperature in step (3) is 25±2°C for 7 to 15 days; the illumination time is 16 h / d, and the illumination intensity is 2000 to 3000 lx.

[0018] The present invention also provides a method for cultivating apricot-plum hybrid seedlings, comprising transferring the test tube seedlings obtained by the embryo rescue method to a greenhouse for cultivation, removing the bottle seedlings after 5 days and transferring them to a substrate for further cultivation in the greenhouse to obtain seedlings, and then transplanting the seedlings to a field.

[0019] In a specific embodiment of the present invention, the matrix includes a mixture of yellow mud and perlite, wherein the mass ratio of the yellow mud to the perlite is 3:1.

[0020] Beneficial effects: The present invention provides a culture medium combination for obtaining apricot-plum hybrid seedlings using an embryo rescue method, wherein the culture medium covers the main culture medium required from embryo germination to adventitious bud differentiation, and the content of each culture medium is optimized. The present invention also provides an embryo rescue method used in conjunction with the culture medium combination, which can effectively promote the continued growth of apricot-plum hybrid embryos under in vitro conditions, improve the embryo germination rate, provide raw materials for obtaining more hybrid offspring, greatly increase the base number of embryo rescue seedlings of apricot-plum hybrid offspring, and the method is highly operable and easy to master. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The young fruits of the “Weidi” variety of apricot plum were picked 62 days after pollination;

[0022] Figure 2 These are the seeds of Wei Di, the top picture shows the seeds with core, and the bottom picture shows the seeds without skin.

[0023] Figure 3 Seed and embryo diagram of Wei Di;

[0024] Figure 4 These are diagrams of embryo development on days 3, 5, and 10 of induction;

[0025] Figure 5 This is a detailed picture of the embryo on the 10th day of induction;

[0026] Figure 6 This is a detailed picture of callus induction one month after induction;

[0027] Figure 7 This is the result of bud induction. DETAILED DESCRIPTION

[0028] The present invention provides a culture medium combination for obtaining apricot-plum hybrid seedlings by using an embryo rescue method, comprising an embryo germination culture medium, a callus induction culture medium and an adventitious bud differentiation culture medium;

[0029] The embryo germination medium uses MS medium as the basic medium and also includes 1-2 mg / L 6-BA, 0.5-1 mg / L IAA and 20-50 g / L sucrose;

[0030] The callus induction medium uses MS medium as the basic medium and also includes 2 mg / L 6-BA and 0.5 mg / L NAA;

[0031] The adventitious bud differentiation medium uses MS medium as the basic medium and further comprises 0.5 mg / L NAA and 0.5-2.0 mg / L 6-BA.

[0032] The main components of the MS culture medium of the present invention include components with the following concentrations: NH4NO3 1650 mg / L, CaCl2 332.2 mg / L, MgSO4 180.7 mg / L, KNO3 1900 mg / L, KH2PO4 170 mg / L, H3BO3 6.2 mg / L, CoCl2·6H2O 0.025 mg / L, CuSO4·5H2O 0.025 mg / L, FeNaEDTA 36.7 mg / L, MnSO4·H2O 16.9 mg / L, Na2MoO4·2H2O 0.25 mg / L, KI 0.83 mg / L and ZnSO4·7H2O 8.6 mg / L, and the pH value is 5.8.

[0033] In a specific embodiment of the present invention, the culture medium combination also includes MS culture medium.

[0034] The present invention also provides a method for obtaining embryo rescue of apricot-plum hybrid seedlings, comprising the following steps: (1) after cross-pollination of apricot-plum varieties, picking fruits at least 60 days after pollination, removing immature embryos after disinfection, and inoculating the immature embryos onto an embryo germination medium in the above-mentioned culture medium combination to induce germination of the immature embryos;

[0035] (2) The germinated embryos are inoculated onto MS medium and cultured for a period of time, and then transferred to callus induction medium to induce callus;

[0036] (3) The callus tissue is inoculated onto an adventitious bud differentiation medium to obtain test tube plantlets.

[0037] In a specific embodiment of the present invention, the fruits of the hybrid pollination of apricot and plum varieties are picked, which can be fruits about 60 days after pollination, such as fruits picked 62 days after artificial pollination in one embodiment. The present invention removes the young embryo from the fruit, and the pulp can be peeled off first, the seeds can be taken out by knocking open the core, and the seeds are disinfected. The disinfection referred to in the embodiment includes first washing with sterile water, and then soaking with a 70% alcohol aqueous solution by volume, rinsing with sterile water, soaking with mercuric chloride and rinsing with sterile water in sequence; such as taking out young seeds, rinsing the young seeds with sterile water 3 to 4 times, then soaking with 70% alcohol for 8 to 10 seconds, rinsing with sterile water 2 to 3 times, disinfecting with mercuric chloride for 8 minutes, and then rinsing with sterile water 3 to 4 times to complete the disinfection. Wherein, the concentration of the mercuric chloride solution can be 0.1%.

[0038] The present invention separates embryos from sterilized seeds, such as carefully taking out young embryos with a dissecting needle in the embodiment, and inoculating them on an embryo germination medium for culture to induce young embryo germination. In a specific embodiment of the present invention, the temperature for inducing young embryo germination in step (1) is 25±2°C, the time is 25-30 days, the illumination time is 16h / d, and the illumination intensity is 2000-3000lx.

[0039] In one embodiment of the present invention, after embryo germination, it is peeled from the endosperm or callus, transferred to MS medium for culture for 30 days, and then transferred to callus medium at a culture temperature of 25±2°C. In one embodiment of the present invention, dark culture is performed on MS medium for 30 days. In one embodiment of the present invention, callus induction is dark culture at a culture temperature of 25±2°C, callus induction is a cycle of 10 days (replacement of culture medium), and the total induction period is 30 to 40 days.

[0040] The MS culture medium used in the present invention contains minerals, vitamins, amino acids, etc. required for plant growth, and can provide necessary nutrients for the growth of plant tissues; regulate hormone balance: MS culture medium usually adds auxin (such as 2,4-D) and cytokinin (such as 6-BA) to regulate the division and differentiation of plant cells and promote the formation of callus; maintain osmotic pressure and pH: appropriate osmotic pressure and pH value help maintain the stable state of cells during the culture process, which is beneficial to cell growth and metabolism; support asexual reproduction: callus can be further differentiated into plants for plant cloning or gene improvement research; the effectiveness of MS culture medium mainly depends on its components and hormone ratio.

[0041] The present invention transfers the callus tissue to an adventitious bud differentiation culture medium, wherein the culture temperature is 25±2°C, the culture time is 7-15 days, the illumination time is 16h / d, and the illumination intensity is 2000-3000lx.

[0042] The present invention also provides a method for cultivating apricot-plum hybrid seedlings, comprising transferring the test tube seedlings obtained by the embryo rescue method to a greenhouse for cultivation, removing the bottle seedlings after 5 days and transferring them to a substrate for further cultivation in the greenhouse to obtain seedlings, and then transplanting the seedlings to a field.

[0043] In a specific embodiment of the present invention, the bottle seedlings are removed, the culture medium is washed, and the medium is soaked in 70% carbendazim and 1000 times fungicide for 10 minutes before being transplanted into the matrix. The matrix comprises a mixture of yellow mud and perlite, wherein the mass ratio of yellow mud to perlite is 3:1. In a specific embodiment of the present invention, after the test tube seedlings are transplanted into the matrix, they are covered to keep moisture, and then removed after 10 days, and then transplanted into the field after 2 months.

[0044] To further illustrate the present invention, a culture medium and method for obtaining apricot-plum hybrid seedlings by embryo rescue provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] Inoculation sampling: Pick the pollinated Figure 1 The 62-day-old "Weidi" variety of apricot plum young fruits shown in the figure were disinfected by first peeling off the flesh, carefully cracking the core, and taking out the young seeds ( Figure 2 ). Rinse the young seeds with sterile water 3-4 times, soak them in 70% alcohol for 8-10 seconds, and rinse them with sterile water 2-3 times. Sterilize them with mercuric chloride for 8 minutes, and then rinse them with sterile water 3-4 times. Carefully remove the young embryos with a scalpel ( Figure 3 ), inoculated into culture medium for cultivation, light intensity 2000~3000lx 16h / d25℃.

[0047] The embryo germination medium is shown in Table 1, comprising: MS medium, sucrose 30 g / L, agar 6.5 g / L;

[0048] Table 1 Types and concentrations of plant hormones in embryo germination medium

[0049]

[0050] The results of embryo germination are shown in Table 2. When 0.5 mg / L 6-BA and 0.2 mg / L IAA were added to MS medium, germination was completed within 15 days ( Figure 4 and Figure 5 ), the germination rate is as high as 77%.

[0051] Table 2 Induction effect of embryo germination medium

[0052] serial number Number of embryos inoculated Germination days / d Germination rate / % <![CDATA[M0]]> 10 25 40 <![CDATA[M1]]> 10 15 77 <![CDATA[M2]]> 10 13 56 <![CDATA[M3]]> 10 11 45

[0053] Callus induction: After embryo germination, the embryos were separated from the endosperm or callus, transferred to MS hormone-free medium and cultured for 30 days, then transferred to callus medium and cultured in the dark at 25±2℃ ( Figure 6 ).

[0054] The concentration of growth regulator in callus induction medium was 2 mg·L -1 6-BA and 0.2mg·L -1 NAA.

[0055] Bud differentiation culture: transfer the callus tissue to the adventitious bud differentiation medium, culture at 25±2℃, and culture for 7 to 15 days to obtain test tube seedlings ( Figure 7 ).

[0056] The concentration of growth regulator in callus induction medium was 1.5 mg·L -1 6-BA and 0.5mg·L -1 NAA.

[0057] Transplanting: First, transfer the test tube seedlings to the greenhouse for cultivation. After 5 days, remove the bottle seedlings, wash the culture medium, soak them in 70% carbendazim 1000 times liquid fungicide for 10 minutes, and then transplant them into small flower pots filled with yellow soil and perlite (m yellow soil: m perlite = 3:1). Put the small flower pots into a basin of water and soak them thoroughly, and cover them with transparent plastic disposable cups to keep them moist. After a week, they will gradually open up, and after 10 days, all transparent plastic cups will be removed. Water, spray foliar fertilizer solution, and prevent and control diseases and insect pests in time as needed. After 2 months, move the transplanted seedlings to the field. Avoid transplanting in the hot summer season and direct sunlight, and provide appropriate shade in the early stage after transplanting.

[0058] The seedlings are hardened under an 80% shade net, and the survival rate can reach 85% 25 days after transplanting.

[0059] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A culture medium combination for obtaining apricot-plum hybrid seedlings using an embryo rescue method, characterized in that: It includes embryo germination medium, callus induction medium and adventitious bud differentiation medium; The embryo germination medium uses MS medium as the basic medium and also includes 1-2 mg / L 6-BA, 0.5-1 mg / L IAA and 20-50 g / L sucrose; The callus induction medium uses MS medium as the basic medium and also includes 2 mg / L 6-BA and 0.5 mg / L NAA; The adventitious bud differentiation medium uses MS medium as the basic medium and further comprises 0.5 mg / L NAA and 0.5-2.0 mg / L 6-BA.

2. The culture medium combination according to claim 1, characterized in that: MS medium is also included.

3. A method for obtaining embryo rescue of apricot-plum hybrid seedlings, characterized in that: The following steps are involved: (1) After cross pollination of apricot and plum varieties, fruits at least 60 days after pollination are picked, and immature embryos are removed after disinfection, and the immature embryos are inoculated onto an embryo germination medium in the medium combination of claim 1 or 2 to induce germination of the immature embryos; (2) The germinated embryos are inoculated onto MS medium and cultured for a period of time, and then transferred to callus induction medium to induce callus; (3) The callus tissue is inoculated onto an adventitious bud differentiation medium to obtain test tube plantlets.

4. The embryo rescue method according to claim 3, characterized in that: The disinfection in step (1) comprises washing the seeds taken from the fruit with sterile water, and then soaking them in a 70% alcohol aqueous solution by volume, rinsing them with sterile water, soaking them in mercuric chloride, and rinsing them with sterile water.

5. The embryo rescue method according to claim 3, characterized in that: The temperature for inducing embryo germination in step (1) is 25±2°C for 25 to 30 days; the illumination time is 16h / d and the illumination intensity is 2000 to 3000lx.

6. The embryo rescue method according to claim 3, characterized in that: Step (2) is to culture in the dark on MS medium, and the dark culture time is 30 days.

7. The embryo rescue method according to claim 3 or 6, characterized in that: The callus induction in step (2) is carried out in dark culture at a temperature of 25±2° C. for 30 to 40 days.

8. The embryo rescue method according to claim 3, characterized in that: The culture temperature in step (3) is 25±2°C for 7 to 15 days; the illumination time is 16 h / d, and the illumination intensity is 2000 to 3000 lx.

9. A method for cultivating apricot-plum hybrid seedlings, characterized in that: The method comprises transferring the test tube seedlings obtained by the embryo rescue method according to any one of claims 3 to 8 to a greenhouse for cultivation, removing the bottle seedlings after 5 days and transferring them to a substrate for further cultivation in the greenhouse to obtain seedlings, and then transplanting the seedlings to the field.

10. The cultivation method according to claim 9, characterized in that: The matrix includes a mixture of yellow mud and perlite, wherein the mass ratio of the yellow mud to the perlite is 3:1.

Images