Pear somatic embryogenesis method

Abstract

The invention belongs to the technical field of biology, and particularly relates to a pear somatic embryogenesis method. Comprising the following steps: collecting fruits and obtaining explants; inducing somatic embryogenesis by taking immature zygotic embryos as explants; taking the immature zygotic embryo as an explant to induce an embryogenic callus; carrying out subculture and embryogenic maintenance on the embryogenic callus; performing somatic embryogenesis on the embryogenic callus; seedling strengthening and rooting culture of regenerated plants; and acclimatizing and transplanting. The two somatic embryo induction ways taking the immature zygotic embryo and the embryonic callus as the explants are high in regeneration efficiency which can reach 100%, good in hereditary stability, good in universality and short in induction time from the explants to the regenerated plants, and are an efficient way for in-vitro rapid propagation of pears; meanwhile, the genetic transformation efficiency of pears can be improved to a certain extent, the genetic stability of transgenic plants is enhanced, and the occurrence probability of chimeras of the transgenic plants is reduced.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for pear somatic cell embryogenesis. Background Art

[0002] Pear is the third largest fruit tree variety in my country, with rich variety resources and high economic value. At present, the traditional sexual hybridization method is mainly used to select new varieties of pear. However, as a woody plant with high genetic heterozygosity, self-incompatibility and long juvenile period, it is quite difficult to carry out genetic improvement through conventional hybridization breeding. Compared with traditional pear genetic improvement, genetic engineering breeding is a more accurate and time-saving method. It can select specific traits and introduce genes related to target traits into pear plants without a large number of gene recombination. It can also avoid the interference of juvenile period to a certain extent, and finally obtain the required transgenic materials. At the same time, with the success of pear genome sequencing, the research on the verification of pear gene function has also been accelerated. Therefore, the establishment and optimization of an efficient genetic transformation system for pear is a prerequisite for verifying gene function through homologous transformation and obtaining new transgenic lines.

[0003] The precursor condition of an efficient genetic transformation system is a stable and efficient regeneration system. The pathways of plant regeneration are generally divided into organogenesis pathways and somatic embryogenesis pathways. At present, the research on pear regeneration mainly focuses on the organogenesis pathway. Although there are reports of 100% regeneration efficiency for autumn pears and western pears, the pear organogenesis pathway is genotype-dependent, and the regeneration efficiency of many pears is low, which cannot meet the requirements of genetic engineering breeding and molecular identification of gene functions. In addition, the organogenesis pathway is mostly an indirect pathway through callus tissue. The induced adventitious buds originate from multiple cells or cell clusters, with poor genetic stability. The transgenic materials obtained through genetic transformation are mostly chimeras, and the transformation efficiency is not high. Compared with the organogenesis pathway, embryoids mostly originate from single cells or small cell clusters, with a lower frequency of cell mutations, and the genetic stability of transgenic plants obtained through genetic transformation is better. At the same time, the number of embryoids induced by somatic cells is greater than the number of adventitious buds formed by organogenesis, and a large number of secondary embryoids will be induced under suitable conditions, so it has a higher proliferation efficiency and a higher genetic transformation efficiency. Moreover, the embryoid development process is similar to that of the zygotic embryo, with a complete structure, similar to the seed structure, and a high seedling rate. Therefore, the embryoid is also a good material for artificial seeds. After being made into artificial seeds, it is easy to store and transport. It can be seen that regeneration through somatic embryo regeneration is a very attractive way for pear in vitro rapid propagation and genetic transformation.

[0004] There are many factors that affect somatic embryogenesis, including genotype, explant type and physiological state, culture medium type and composition, culture environment, etc. Therefore, it is difficult to screen suitable explants and culture medium formulas and induce somatic embryogenesis. Therefore, there are few reports on the pathway of pear somatic embryogenesis, and there is no universal somatic embryo induction method. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for pear somatic embryogenesis. The method is simple to operate and has good universality.

[0006] The technical solution adopted by the present invention is as follows:

[0007] (1) Fruit collection and explant acquisition

[0008] Pear fruits were collected 30-60 days after flowering, and after surface disinfection, the fruits were cut open, immature seeds were taken out, the seed coats were opened, and immature zygotic embryos were squeezed out as explants;

[0009] (2) Inducing somatic embryogenesis using immature zygotic embryos as explants

[0010] The explant obtained in step (1) is inoculated on a MS medium with a pH of 5.82, and cultured at 25° C. in the dark for 20-25 days, and a mature somatic embryo is formed from a globular embryo, through a heart-shaped embryo and a torpedo-shaped embryo development process. The somatic embryo can germinate and form a regenerated bud after 30-35 days;

[0011] (3) Inducing embryonic callus using immature zygotic embryos as explants

[0012] The explant obtained in step (1) was inoculated on MS medium with a pH of 5.82, and cultured at 25° C. in the dark to obtain embryonic callus after 30 days;

[0013] (4) Subculture and embryonic maintenance of embryonic callus

[0014] Subculturing and proliferating the embryonic callus obtained in step (3) on MS medium with a pH of 5.82 at 25° C. and in the dark to maintain the embryonic nature of the callus;

[0015] (5) Somatic Embryogenesis of Embryogenic Callus

[0016] Transferring the embryonic callus obtained in steps (3) and (4) to MS medium with a pH of 5.82, culturing at 25° C. in the dark, and inducing the formation and development of somatic embryos;

[0017] (6) Rooting and seedling cultivation of regenerated plants

[0018] Placing the somatic embryonic regeneration plantlets obtained in steps (2) and (5) in MS rooting medium with a pH of 5.82, and culturing them for 30 days at a culture temperature of 25° C., a light intensity of 1500 lux, and a light duration of 16 h / d to obtain complete self-rooted seedlings;

[0019] (7) Hardening and transplanting of seedlings.

[0020] Furthermore, in step (2), the formula of the MS medium is: 1.0-3.0 mg / L NAA, 0.2-1.5 mg / L TDZ, 30 g / L sucrose, and 7 g / L agar.

[0021] Furthermore, in step (3), the formula of the MS medium is: 1.0-2.0 mg / L 2,4-D, 0.5-1.0 mg / L KT or 6-BA, 30 g / L sucrose, and 7 g / L agar.

[0022] Furthermore, in step (4), the formula of the MS medium is: 1.0-2.0 mg / L 2,4-D, 1.0 mg / L 6-BA, 30 g / L sucrose, and 7 g / L agar.

[0023] Furthermore, the formula of the MS medium in step (5) is: 1.0-3.0 mg / L NAA, 0.2-1.5 mg / L TDZ, 30 g / L sucrose, and 7 g / L agar.

[0024] Furthermore, in step (6), the formula of the MS rooting medium is: 0.05-0.2 mg / L IBA, 30 g / L sucrose, and 7 g / L agar.

[0025] Furthermore, the specific steps in step (7) are: after hardening the complete self-rooted seedlings obtained in step (6) in a room at a temperature of 25°C for 2-5 days, take out the seedlings, wash the root culture medium, transplant them into a matrix with a vermiculite: straw carbon ratio of 1:1, and place them in an incubator with a temperature of 25°C, a light intensity of 1000 lux, and a light time of 16h / d.

[0026] The beneficial effects of the present invention are as follows: 1) the present invention uses immature embryos 30-60 days after pear flowering as explants, and can induce somatic embryos and regenerate plants in a relatively short period of time (20-25 days); at the same time, using immature embryos 30-60 days after pear flowering as explants, embryonic callus can be induced and the embryonic nature of the callus can be maintained through subculture proliferation, and the embryonic callus can be transferred to a somatic embryo induction medium to induce somatic embryos and regenerate plants; the regenerated plants can be transplanted and survive normally after rooting culture;

[0027] 2) The two somatic embryo induction pathways have high regeneration efficiency, which can reach 100%, good genetic stability and universality, which can help to improve the efficiency of pear genetic transformation to a certain extent, enhance the genetic stability of transgenic plants, and reduce the occurrence rate of chimeras in transgenic plants;

[0028] 3) The present invention uses NAA and TDZ to induce somatic embryos in 20-25 days, and the somatic embryos can germinate to form regenerated buds in 30-35 days. The induction time from explants to regenerated plants is short. This technology can significantly shorten the time for pear clone propagation and genetic transformation, and improve efficiency;

[0029] 4) The present invention uses immature embryos as explants, uses 2,4-D to induce embryonic callus, and simultaneously screens out a suitable formula that can allow the embryonic callus to be subcultured for a long time without losing its embryonic nature. Finally, the obtained somatic embryo induction formula is used to induce somatic embryos from the embryonic callus. This technology solves the problem that when using embryos as explants to induce plant regeneration, primary culture is required for each operation, and the genetic background of the regenerated plants is not uniform when a large number of inductions are performed. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Embryogenic callus diagram

[0031] In the figure, the upper part shows callus tissue induced on the medium containing 2,4-D; the lower part shows callus tissue observed by transmission electron microscope;

[0032] Figure 2 Diagram showing the pathway of somatic embryogenesis in pear

[0033] In the figure, A. globular embryo, B. heart-shaped embryo, C. torpedo-shaped embryo, D. cotyledon-shaped embryo, and E. somatic embryo germination. DETAILED DESCRIPTION

[0034] Example 1 Effects of different auxins on pear somatic embryogenesis

[0035] (1) Pear fruits were collected 60 days after flowering, brought back to the laboratory, rinsed with tap water, and disinfected with 75% alcohol (30S) and 2% sodium hypochlorite (10min) in a clean bench. They were then rinsed 3-5 times with sterile water, the fruits were cut open, the immature seeds were taken out, the seed coats were opened with a dissecting needle, and the immature zygotic embryos were squeezed out as explants;

[0036] (2) The above explants were inoculated on MS medium containing 0.5 mg / L KT, different concentrations of 2,4-D, NAA, or IAA (1.0-3.0 mg / L), 30 g / L sucrose, 7 g / L agar, pH = 5.82, and cultured at 25°C in the dark. After 30 days, the growth and development of each explant was observed.

[0037] like Figure 1 As shown, the explants grown on 2,4-D and KT medium obtained a large amount of yellow or milky white, granular callus tissue. Under transmission electron microscopy, the cell contents were rich and the embryonic state was good, but continued culture could not form somatic embryos.

[0038] Explants grown on NAA and KT medium can induce somatic embryos. After experiencing globular embryos, heart-shaped embryos, and torpedo-shaped embryos, somatic embryos germinate to obtain regenerated plantlets ( Figure 2 ); Under the condition of 1.0-3.0 mg / L NAA, the somatic embryo induction rate can reach 34-40% (Table 1).

[0039] Under IAA and KT medium culture, a small amount of somatic embryos were formed in the explants, the somatic embryo induction rate was no higher than 21.88%, and the embryonic callus induction rate was low (Table 1). It can be seen that the MS medium containing 1.0-3.0 mg / L NAA, 0.5 mg / L KT, 30 g / L sucrose, 7 g / L agar, pH = 5.82 is a more suitable medium for inducing immature pear embryos to form embryoids, and the MS medium containing 1.0-2.0 mg / L 2,4-D, 0.5-1.0 mg / L KT or 6-BA, 30 g / L sucrose, 7 g / L agar, pH = 5.82 is a more suitable medium for inducing embryonic callus.

[0040] Table 1 Induction of embryoids by different auxin concentrations

[0041]

[0042] Example 2 Effect of explant maturity on pear somatic embryogenesis

[0043] (1) Pear fruits were collected 30, 60, 90, and 110 days after anthesis and brought back to the laboratory. After being rinsed with tap water, the surface was disinfected with 75% alcohol (30 seconds) and 2% sodium hypochlorite (10 minutes) in a clean bench and then rinsed 3-5 times with sterile water. The fruits were cut open, and the immature seeds were taken out. The seed coat was picked open with a dissecting needle, and the immature zygotic embryos were squeezed out as explants.

[0044] (2) The above explants were inoculated on MS medium containing 0.5 mg / L KT, 1.0 mg / L NAA, 30 g / L sucrose, 7 g / L agar, pH = 5.82, and cultured at 25°C in the dark, and then the somatic embryo formation of each explant was counted. As shown in Table 3, under the same culture conditions, the induction rate of somatic embryos was higher when the less mature zygotic embryos (30d and 60d) were used as explants, and the induction coefficient was also the highest (Table 2).

[0045] Table 2 Induction of embryoids by zygotic embryos of different maturity

[0046]

[0047] Example 3 Effects of different cytokinins on pear somatic embryogenesis

[0048] (1) Pear fruits were collected 60 days after flowering and brought back to the laboratory. After being rinsed with tap water, the surface was disinfected with 75% alcohol (30S) and 2% sodium hypochlorite (10min) in a clean bench and then rinsed with sterile water for 3-5 times. The fruits were cut open, and the immature seeds were taken out. The seed coat was picked open with a dissecting needle, and the immature zygotic embryos were squeezed out as explants.

[0049] (2) The above explants were inoculated on MS medium containing 1.0 mg / L NAA, different concentrations of TDZ, 6-BA, KT or ZT (0.2-1.5 mg / L), 30 g / L sucrose, 7 g / L agar, pH = 5.82, and cultured at 25°C in the dark. The growth and development of each explant was observed after 30 days. As shown in Table 3, the explants grown on TDZ medium can induce a large number of somatic embryos. After experiencing globular embryos, heart-shaped embryos, and torpedo-shaped embryos, somatic embryos germinate to obtain regenerated plantlets; and under the treatment of 1.5 mg / L TDZ, the somatic embryo induction rate can be as high as 100%, and the induction coefficient is 13.76. A certain amount of somatic embryos can also be induced on 6-BA, KT and ZT medium, but the efficiency is less than 41%.

[0050] Table 3 Effects of different cytokinin concentrations on somatic embryo induction

[0051]

[0052]

Claims

1. A method for pear somatic embryogenesis, characterized in that: The following steps are involved: (1) Fruit collection and explant acquisition Pear fruits were collected 30-60 days after flowering, and after surface disinfection, the fruits were cut open, immature seeds were taken out, the seed coats were opened, and immature zygotic embryos were squeezed out as explants; (2) Inducing somatic embryogenesis using immature zygotic embryos as explants The explant obtained in step (1) is inoculated on a MS medium with a pH of 5.82, and cultured at 25° C. in the dark for 20-25 days, and a mature somatic embryo is formed from a globular embryo, through a heart-shaped embryo and a torpedo-shaped embryo development process. The somatic embryo can germinate and form a regenerated bud after 30-35 days; (3) Inducing embryonic callus using immature zygotic embryos as explants The explant obtained in step (1) was inoculated on MS medium with a pH of 5.82, and cultured at 25° C. in the dark to obtain embryonic callus after 30 days; (4) Subculture and embryonic maintenance of embryonic callus Subculturing and proliferating the embryonic callus obtained in step (3) on MS medium with a pH of 5.82 at 25° C. and in the dark to maintain the embryonic nature of the callus; (5) Somatic Embryogenesis of Embryogenic Callus Transferring the embryonic callus obtained in steps (3) and (4) to MS medium with a pH of 5.82, culturing at 25° C. in the dark, and inducing the formation and development of somatic embryos; (6) Rooting and seedling cultivation of regenerated plants Placing the somatic embryonic regeneration plantlets obtained in steps (2) and (5) in MS rooting medium with a pH of 5.82, and culturing them for 30 days at a culture temperature of 25° C., a light intensity of 1500 lux, and a light duration of 16 h / d to obtain complete self-rooted seedlings; (7) Hardening and transplanting of seedlings.

2. A method for pear somatic embryogenesis as claimed in claim 1, characterized in that: The formula of MS medium in step (2) is: 1.0-3.0 mg / L NAA, 0.2-1.5 mg / L TDZ, 30 g / L sucrose, and 7 g / L agar.

3. A method for pear somatic embryogenesis as claimed in claim 1, characterized in that: The formula of MS medium in step (3) is: 1.0-2.0 mg / L 2,4-D, 0.5-1.0 mg / L KT or 6-BA, 30 g / L sucrose, and 7 g / L agar.

4. The method for pear somatic embryogenesis according to claim 1, wherein: The formula of MS medium in step (4) is: 1.0-2.0 mg / L 2,4-D, 1.0 mg / L 6-BA, 30 g / L sucrose, and 7 g / L agar.

5. The method for pear somatic embryogenesis according to claim 1, wherein: Step (5) The formula of MS culture medium is: containing 1.0-3.0 mg / L NAA, 0.2-1.5 mg / L TDZ, 30 g / L sucrose, and 7 g / L agar.

6. The method for pear somatic embryogenesis according to claim 1, wherein: The formula of MS rooting medium in step (6) is: 0.05-0.2 mg / L IBA, 30 g / L sucrose, and 7 g / L agar.

7. The method for pear somatic embryogenesis according to claim 1, wherein: The specific steps in step (7) are as follows: after hardening the complete self-rooted seedlings obtained in step (6) in a room at a temperature of 25°C for 2-5 days, the seedlings are taken out, the root culture medium is washed, and the seedlings are transplanted into a matrix of vermiculite: straw carbon in a ratio of 1:1, and placed in an incubator with a temperature of 25°C, a light intensity of 1000 lux, and a light time of 16h / d for cultivation.

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