A method of pear somatic embryogenesis

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a method for somatic embryogenesis of pear. The method comprises the following steps: fruit collection and explant obtaining; inducing somatic embryogenesis by using immature zygotic embryo as explant; inducing embryogenic callus by using immature zygotic embryo as explant; subculture of embryogenic callus and embryogenic maintenance; somatic embryogenesis of embryogenic callus; strong seedling and rooting culture of regenerated plant; and seedling raising and transplanting. The two somatic embryogenesis induction pathways of the present application, which use immature zygotic embryo and embryogenic callus as explants, have high regeneration efficiency, good genetic stability, good universality, short induction time from explant to regenerated plant, and are a high-efficiency pathway for in vitro rapid propagation of pear. Meanwhile, the method can help to improve the genetic transformation efficiency of pear, enhance the genetic stability of transgenic plants, and reduce the occurrence probability of transgenic plant chimeras.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for somatic embryogenesis of Pyrus. BACKGROUND

[0002] Pear is the third largest fruit tree species in China, with rich variety resources and high economic value. At present, new varieties of pear are mainly selected by traditional sexual crossing. However, as a woody plant with high genetic heterozygosity, self-incompatibility and long juvenile period, it is quite difficult to carry out genetic improvement by conventional crossing breeding. Compared with traditional pear genetic improvement, genetic engineering breeding is a more accurate and time-saving method, which can select specific traits and introduce genes related to target traits into pear plants, without large-scale gene recombination and interference of juvenile period, so as to obtain the desired transgenic materials. Meanwhile, with the success of pear genome sequencing, the research on pear gene function verification is also accelerated. Therefore, the establishment and optimization of high-efficiency genetic transformation system of pear is the premise of verifying gene function by homologous transformation and obtaining transgenic new lines.

[0003] The precursor condition of high-efficiency genetic transformation system is stable and efficient regeneration system. The plant regeneration pathway is generally divided into organogenesis pathway and somatic embryogenesis pathway. At present, the research on pear regeneration mainly focuses on organogenesis pathway. Although there are related reports that Pyrus ussuriensis and Pyrus communis have 100% regeneration efficiency, the pear organogenesis pathway has genotype dependence, and the regeneration efficiency of many pears is low, which cannot meet the requirements of genetic engineering breeding and gene function molecular identification. Moreover, the organogenesis pathway is mostly indirect through callus, the induced adventitious buds originate from multicellular or cell clusters, and the genetic stability is poor, so that the transgenic materials obtained by genetic transformation are mostly chimeras, and the transformation efficiency is not high. Compared with the organogenesis pathway, the somatic embryoid originates from single cell or small cell cluster, the cell variation frequency is low, and the genetic stability of the transgenic plants obtained by genetic transformation is better. At the same time, the number of somatic embryoids induced by somatic cells is more than that of adventitious buds formed by organogenesis, and a large number of secondary somatic embryoids will be induced under suitable conditions, so that the proliferation efficiency is high, and the genetic transformation efficiency is also high. Moreover, the development process of somatic embryoid is similar to zygotic embryo, the structure is complete, similar to the structure of seed, and the seedling rate is high. Therefore, somatic embryoid is also a good material for artificial seed. After the artificial seed is prepared, it is convenient for storage and transportation. It can be seen that the regeneration by somatic embryogenesis pathway is a quite attractive way for pear in vitro rapid propagation and genetic transformation.

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

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

[0006] The technical scheme adopted by the present application is as follows:

[0007] (1) Fruit collection and explant acquisition

[0008] Pear fruits are collected at 30-60 days after flowering, the surface is disinfected, the fruits are cut open, the immature seeds are taken out, the seed coat is picked open, and the immature zygotic embryos are squeezed out as explants;

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

[0010] The explants obtained in step (1) are inoculated on MS medium with pH = 5.82 and cultured at 25°C in the dark for 20-25 days, and mature somatic embryos are formed through the development process of globular embryos, heart-shaped embryos and torpedo-shaped embryos, and the somatic embryos can germinate to form regenerated shoots in 30-35 days;

[0011] (3) Inducing embryogenic callus with immature zygotic embryos as explants

[0012] The explants obtained in step (1) are inoculated on MS medium with pH = 5.82 and cultured at 25°C in the dark, and embryogenic callus is obtained in 30 days;

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

[0014] The embryogenic callus obtained in step (3) is subcultured and proliferated on MS medium with pH = 5.82 and cultured at 25°C in the dark to maintain the embryonic nature of the callus;

[0015] (5) Somatic embryogenesis of embryogenic callus

[0016] The embryogenic callus obtained in steps (3) and (4) is transferred to MS medium with pH = 5.82 and cultured at 25°C in the dark to induce the formation and development of somatic embryos;

[0017] (6) Rooting culture of strong seedlings of regenerated plants

[0018] The somatic embryo regenerated plantlets obtained in steps (2) and (5) were placed in MS rooting medium with pH = 5.82 and cultured for 30 days at a culture temperature of 25℃, a light intensity of 1500 lux, and a light duration of 16 h / d to obtain complete self-rooted seedlings.

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

[0020] Furthermore, the MS medium formulation 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.

[0021] Furthermore, the MS medium formulation 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.

[0022] Furthermore, the MS medium formulation 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.

[0023] Furthermore, the MS medium formulation for step (5) 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.

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

[0025] Further, the specific steps in step (7) are as follows: after the complete self-rooted seedlings obtained in step (6) are hardened in an indoor environment at a temperature of 25℃ for 2-5 days, the seedlings are taken out, the root culture medium is washed off, and they are transplanted into a substrate of vermiculite: peat moss in a 1:1 ratio and placed in an incubator at a temperature of 25℃, a light intensity of 1000 lux, and a light duration of 16 h / d.

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

[0027] 2) Both somatic cell embryo induction pathways have high regeneration efficiency, reaching 100%, good genetic stability, and good universality. They can help improve the genetic transformation efficiency of pears to a certain extent, enhance the genetic stability of transgenic plants, and reduce the probability of chimerism in transgenic plants.

[0028] 3) This 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 explant to regenerated plant is relatively short. This technology can significantly shorten the time for asexual propagation and genetic transformation of pear lines and improve efficiency.

[0029] 4) This invention uses immature embryos as explants and 2,4-D to induce embryogenic callus. At the same time, a suitable formula is screened to allow long-term subculturing of embryogenic callus without losing its embryogenicity. Finally, somatic embryos are induced from embryogenic callus using the obtained somatic embryo induction formula. This technology solves the problem that when using embryos as explants to induce plant regeneration, it is necessary to perform primary culture for each operation, and the genetic background of regenerated plants is not uniform when inducing a large number of plants. Attached Figure Description

[0030] Figure 1 Image showing embryonic callus

[0031] Top image: Callus induced on medium containing 2,4-D; Bottom image: Transmission electron microscopy observation of the callus.

[0032] Figure 2 Diagram illustrating the pear somatic embryogenesis pathway.

[0033] In the diagram, A. spherical embryo, B. heart-shaped embryo, C. torpedo-shaped embryo, D. cotyledon-shaped embryo, and E. somatic embryo germination. Detailed Implementation

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

[0035] (1) Collect pear fruits 60 days after flowering, bring them back to the laboratory, rinse them with tap water, disinfect them with 75% alcohol (30s) and 2% sodium hypochlorite (10min) in a clean bench, rinse them with sterile water 3-5 times, cut the fruits open, take out the immature seeds, use a dissecting needle to pick open the seed coat, squeeze out the immature zygote embryo as an explant.

[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, and pH=5.82. They were cultured at 25°C in the dark and observed for 30 days.

[0037] like Figure 1 As shown, explants grown on 2,4-D and KT media yielded a large amount of yellow or milky white granular callus tissue. Under transmission electron microscopy, the cells were rich in contents and had good embryogenicity, but continued culture could not form somatic embryos.

[0038] Explants grown on NAA and KT media can induce somatic embryos, which, after undergoing processes of spherical, heart-shaped, and torpedo-shaped embryos, germinate to produce regenerated plantlets. Figure 2 Under conditions of 1.0-3.0 mg / L NAA, the somatic embryo induction rate can reach 34-40% (Table 1).

[0039] Under IAA and KT culture media, a small number of somatic embryos formed from explants, with a somatic embryo induction rate not exceeding 21.88%, and the induction rate of embryogenic callus was low (Table 1). Therefore, MS medium containing 1.0-3.0 mg / L NAA, 0.5 mg / L KT, 30 g / L sucrose, 7 g / L agar, and pH 5.82 is a suitable medium for inducing embryoid formation from immature pear embryos, while 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, and pH 5.82 is a suitable medium for inducing embryogenic 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) Collect pear fruits 30, 60, 90 and 110 days after flowering, bring them back to the laboratory, rinse them with tap water, disinfect them with 75% alcohol (30s) and 2% sodium hypochlorite (10min) in a clean bench, rinse them with sterile water 3-5 times, cut open the fruits, take out the immature seeds, use a dissecting needle to pick open the seed coat, squeeze out the immature zygote embryo as an explant.

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

[0045] Table 2. Induction of embryoids by zygotes of different maturity levels.

[0046]

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

[0048] (1) Collect pear fruits 60 days after flowering, bring them back to the laboratory, rinse them with tap water, disinfect them with 75% alcohol (30s) and 2% sodium hypochlorite (10min) in a clean bench, rinse them with sterile water 3-5 times, cut open the fruits, take out the immature seeds, use a dissecting needle to pick open the seed coat, squeeze out the immature zygote embryo as an explant.

[0049] (2) The explants were inoculated onto MS medium containing 1.0 mg / L NAA, and different concentrations of TDZ, 6-BA, KT or ZT (0.2-1.5 mg / L), 30 g / L sucrose, 7 g / L agar, and pH 5.82. The explants were cultured at 25°C in the dark for 30 days, and their growth and development were observed. As shown in Table 3, explants grown on TDZ medium could induce a large number of somatic embryos. After undergoing globular, heart-shaped, and torpedo-shaped embryos, the somatic embryos germinated to obtain regenerated small plantlets. Under 1.5 mg / L TDZ treatment, the somatic embryo induction rate could reach 100%, with an induction coefficient of 13.76. A certain amount of somatic embryos could also be induced on 6-BA, KT, and ZT media, but the efficiency was 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, Includes the following steps: (1) Fruit collection and explant acquisition Pear fruits were collected 30-60 days after flowering. After surface disinfection, the fruits were cut open, immature seeds were removed, the seed coat was removed, and immature zygotic embryos were squeezed out as explants. (2) Inducing somatic embryogenesis using immature zygote as explants The explants obtained in step (1) were inoculated on MS medium containing 1.0 mg / L NAA, 0.2-1.5 mg / L TDZ, 30 g / L sucrose, 7 g / L agar, and pH=5.

82. They were cultured at 25 ℃ in the dark for 20-25 days. The embryos developed from spherical embryos through the process of heart-shaped embryos and torpedo-shaped embryos to form mature somatic embryos. The somatic embryos could germinate and form regenerated buds in 30-35 days. (3) Inducing embryogenic callus using immature zygotes as explants The explants obtained in step (1) were inoculated on MS medium containing 1.0-2.0 mg / L 2,4-D, 0.5 mg / L KT, 30 g / L sucrose, 7 g / L agar, and pH=5.82, and cultured at 25 ℃ in the dark for 30 days to obtain embryogenic callus. (4) Subculture and preservation of embryogenic callus The embryogenic callus obtained in step (3) was subcultured and proliferated on MS medium containing 1.0-2.0 mg / L 2,4-D, 1.0 mg / L 6-BA, 30 g / L sucrose, 7 g / L agar, pH=5.82, and cultured in the dark at 25 ℃ to maintain the embryogenicity of the callus. (5) Somatic embryogenesis of embryonic callus The embryogenic callus obtained in steps (3) and (4) was transferred to MS medium containing 1.0-3.0 mg / L NAA, 0.2-1.5 mg / LTDZ, 30 g / L sucrose, 7 g / L agar, pH=5.82, and cultured at 25 ℃ in the dark to induce the formation and development of somatic embryos. (6) Rooting culture of regenerated plantlets The somatic embryo regenerated plantlets obtained in steps (2) and (5) were placed in MS rooting medium containing 0.05-0.2 mg / L IBA, 30 g / L sucrose, 7 g / L agar, and pH=5.

82. They were cultured for 30 days at a temperature of 25℃, a light intensity of 1500 lux, and a light duration of 16 h / d to obtain complete self-rooted seedlings. (7) Hardening off seedlings and transplanting.

2. The method for pear somatic embryogenesis as described in claim 1, characterized in that: The specific steps in step (7) are as follows: After the complete self-rooted seedlings obtained in step (6) are hardened in an indoor environment at a temperature of 25℃ for 2-5 days, the seedlings are taken out, the root culture medium is washed off, and they are transplanted into a substrate of vermiculite: peat moss in a 1:1 ratio and placed in an incubator at a temperature of 25℃, a light intensity of 1000 lux, and a light duration of 16 h / d.

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