A method for constructing an efficient somatic embryo regeneration system of carnation

Abstract

The present invention discloses a method for constructing an efficient somatic embryo regeneration system of carnation, which includes steps such as seed germination, induction of callus, induction of embryogenic callus, differentiation of embryogenic callus, rooting culture, etc. The efficient regeneration system constructed by the method of the present invention realizes rapid, stable and efficient breeding of carnation.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for constructing a carnation somatic embryo efficient regeneration system. Background Art

[0002] Carnation ( Dianthus caryophyllus L.), also known as carnation, belongs to the Caryophyllaceae ( Caryophyllaceae ) Dianthus Dianthus ), is a perennial herb. Carnation is monoecious, but due to the high degree of double petals in cultivated varieties, the number of stamens is scarce, and most of them are hidden deep inside the petals, and even the stamens of some varieties have been aborted. The existing carnation regeneration technology usually induces callus tissue by cutting different explant materials, and then the callus tissue differentiates and sprouts, roots, and then hardens the seedlings and transplants. This process first requires the explant to be disinfected, but since the explants with strong regeneration ability are usually relatively young and tender, if the disinfection is not thorough during the disinfection process, it is easy to cause contamination, and excessive disinfection may cause the death of young materials. Therefore, sterile seedlings are usually obtained first, and then propagated by cutting. However, after multiple subcultures, the reproduction coefficient of sterile seedlings will decrease, the growth of the plants will weaken, and over time, the plants may have a virus accumulation effect, resulting in a decrease in flowering quality. In addition, the process of cutting explants from sterile seedlings to induce callus tissue and differentiate buds is time-consuming and has a low seedling success rate. Therefore, it is very necessary to study the efficient regeneration technology of carnation. Summary of the invention

[0003] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for constructing a high-efficiency regeneration system of somatic embryos of carnation. The high-efficiency regeneration system constructed by the method of the present invention realizes rapid, stable and efficient breeding of carnation.

[0004] The objective of the present invention is achieved through the following technical solutions:

[0005] A method for constructing a highly efficient regeneration system of carnation somatic embryos comprises the following steps:

[0006] (1) Seed disinfection and sterilization: Dianthus caryophyllus seeds were first soaked in 75% alcohol and then rinsed with sterile water, then soaked in 3% sodium hypochlorite solution and then rinsed with sterile water, and then the moisture on the surface of the seeds was removed and inoculated onto a basic culture medium of MS + 30 g / L sugar + 8 g / L agar, pH = 6 ± 0.2.

[0007] (2) Seed germination: The seeds inoculated on the basal medium were first placed in the dark at 25±2℃ until the seeds began to germinate. The germinated seeds were transferred to the light conditions and cultured at 25±2℃, 16h photoperiod, and 36μmol·m -2 ·s-1 Cultured until carnation seedlings are obtained.

[0008] (3) Induction of callus: Take carnation seedlings, cut off the hypocotyls, and place them on a callus induction medium of MS + 30 g / L sugar + 8 g / L agar + 1 - 5 mg / L 2,4-D + 10 -6 - 10 -8 μmol / L PSK, place in a dark environment at 25 ± 2 °C, and induce and culture until callus is produced.

[0009] (4) Induction of embryogenic callus: Transfer the induced callus to an embryogenic callus induction medium of MS + 30 g / L sugar + 8 g / L agar + 0.5 - 2.5 mg / L 6-BA + 0.1 - 2.0 mg / L NAA, under light conditions, at 25 ± 2 °C, a photoperiod of 16 h, and a light intensity of 36 μmol·m -2 ·s -1 Induce and culture until embryogenic callus is produced.

[0010] (5) Differentiation of embryogenic callus: Chop the induced embryogenic callus and place it in a differentiation medium of MS + 30 g / L sugar + 8 g / L agar + 0.5 - 2.5 mg / L NAA + 0.1 - 2.0 mg / L TDZ + 0.5 - 2.5 mg / L KT, under light conditions, at 25 ± 2 °C, a photoperiod of 16 h, and a light intensity of 36 μmol·m -2 ·s -1 Carry out differentiation culture until somatic embryos differentiate into buds.

[0011] (6) Rooting culture: Separate the cotyledon embryos with well-developed cotyledons from the embryogenic callus, inoculate them in a rooting medium of MS + 30 g / L sucrose + 8 g / L agar, pH = 6 ± 0.2, and culture at a light intensity of 40 μmol·m -2 ·s -1 , a photoperiod of 16 h, and 23 ± 2 °C until roots are formed. After the regenerated seedlings take root, place a substrate with a mass ratio of peat soil to perlite of 2:1 in a plug tray, plant the rooted seedlings in the plug tray, and transplant them after adapting to the natural environment.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] 1. The present invention has established an efficient somatic embryo regeneration system for carnation, with the number of somatic embryo inductions per embryogenic callus reaching more than 1600, providing an efficient genetic transformation system based on somatic embryogenesis for carnation genetic engineering breeding.

[0014] 2. The present invention can achieve the mass propagation of healthy carnation plants, effectively avoid the virus accumulation effect in traditional cuttage propagation, and thus improve the propagation efficiency and seedling quality of asexual reproduction. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Callus induced from the hypocotyl of carnation in Example 2 after 45 days of induction in the callus induction medium of MS + 30 g / L sugar + 8 g / L agar + 4 mg / L 2,4-D + 10 -7 mol / L PSK.

[0016] Figure 2 Callus induced from the cotyledon of carnation in Example 2 after 45 days of induction in the callus induction medium of MS + 30 g / L sugar + 8 g / L agar + 4 mg / L 2,4-D + 10 -7 mol / L PSK.

[0017] Figure 3 Phenotype of the hypocotyl callus of carnation in Example 3 after 40 days of induction culture.

[0018] Figure 4 Phenotype of the cotyledon callus of carnation in Example 3 after 40 days of induction culture.

[0019] Figure 5 Phenotype of the embryogenic callus of the hypocotyl of carnation in Example 3 after 11 days of differentiation culture.

[0020] Figure 6 Phenotype of the embryogenic callus of the hypocotyl of carnation in Example 3 after 30 days of differentiation culture.

[0021] Figure 7 Stereomicroscope image of the seedlings after somatic embryo differentiation of carnation in Example 3.

[0022] Figure 8 Micrograph of paraffin section of the globular embryo of somatic embryo of carnation in Example 3.

[0023] Figure 9 Micrograph of paraffin section of the heart-shaped embryo of somatic embryo of carnation in Example 3.

[0024] Figure 10 Regenerated seedlings of carnation after rooting in Example 1.

[0025] Figure 11 Statistical chart of the differentiation of somatic embryos into buds during the regeneration of carnation using the method described in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited to the embodiments. Example 1

[0027] A method for constructing an efficient somatic embryo regeneration system of carnation, comprising the following steps:

[0028] (1) Disinfection and sterilization of seeds: Carnation seeds are first soaked in 75% alcohol for 30 s, then rinsed 3 times with sterile water, and then soaked in 3% sodium hypochlorite solution for 8 - 10 min. During this process, the beaker is shaken continuously to ensure sufficient disinfection. After that, the disinfectant is poured out, and the seeds are rinsed 3 - 5 times with sterile water. After rinsing, the seeds are blown onto sterile filter paper to absorb the residual moisture on the surface of the seeds, and then inoculated onto the basic medium: MS + 30 g / L sugar + 8 g / L agar, pH = 5.8. Among them, the components of MS are: KNO3 1900 mg / L, NH4NO3 1650 mg / L, KH2PO4 170 mg / L, MgSO4·7H2O 370 mg / L, CaCl2·2H2O 440 mg / L, KI 0.83 mg / L, H3BO3 6.2 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2·6H2O 0.025 mg / L, Na2-EDTA·2H2O 37.3 mg / L, FeSO4·7H2O 27.8 mg / L, inositol 100 mg / L, glycine 2 mg / L, thiamine hydrochloride 0.1 mg / L, pyridoxine hydrochloride 0.5 mg / L, nicotinic acid 0.5 mg / L.

[0029] (2) Seed germination: The seeds inoculated on the basic medium are first placed under dark conditions at 25 ± 2 °C and cultured in the dark for 3 - 5 d. At this time, the seeds have started to germinate. The germinated seeds are transferred to light conditions and cultured at 25 ± 2 °C, a photoperiod of 16 h / 8 h, and a light intensity of 36 μmol·m -2 ·s -1 for 5 - 7 d to obtain carnation seedlings.

[0030] (3) Induction of callus: Take carnation seedlings in a clean bench, cut off the hypocotyls and place them on the callus induction medium of MS + 30 g / L sugar + 8 g / L agar + 4 mg / L 2,4-D + 10 -7 μmol / L PSK, and place them in a dark environment at 25 ± 2 °C for 40 - 50 d to induce the production of callus.

[0031] (4) Induction of embryogenic callus: Transfer the induced callus to an embryogenic callus induction medium of MS + 30 g / L sugar + 8 g / L agar + 1.5 mg / L 6-BA + 0.5 mg / L NAA, and under light conditions, at 25 ± 2 °C, a photoperiod of 16 h / 8 h, and a light intensity of 36 μmol·m -2 ·s -1 Induce and culture for 30 - 40 d until embryogenic callus is produced.

[0032] (5) Differentiation of embryogenic callus: Chop the induced embryogenic callus and place it in a differentiation medium of MS + 30 g / L sugar + 8 g / L agar + 1 mg / L NAA + 0.25 mg / L TDZ + 1 mg / L KT. Under light conditions, at 25 ± 2 °C, a photoperiod of 16 h / 8 h, and a light intensity of 36 μmol·m -2 ·s -1 Conduct differentiation culture for 20 - 30 d until somatic embryos differentiate into buds.

[0033] (6) Rooting culture: In a laminar flow hood, separate the cotyledon embryos with well-developed cotyledons from the embryogenic callus, and inoculate them respectively into a rooting medium of MS + 30 g / L sucrose + 8 g / L agar, pH = 5.8, for rooting culture. Inoculate 6 seedlings per bottle. The culture conditions are a light intensity of 40 μmol·m -2 ·s -1 , a photoperiod of 16 h / 8 h, a temperature of 23 ± 2 °C, and culture for 7 - 14 d. After the regenerated seedlings take root (as Figure 10 shown), place a substrate with a mass ratio of peat soil to perlite of 2:1 in a plug tray. Take the rooted seedlings out of the medium, wash the remaining medium on the roots with pure water, and then plant them in the plug tray. After adapting to the natural environment, transplant them.

[0034] Example 2 - Comparison of the effects of different concentrations of plant growth regulators on the induction of carnation callus

[0035] 1. Comparison of the induction rates of callus with different concentrations of 2,4-D

[0036] In a laminar flow hood, take the carnation seedlings cultured in step (2) of Example 1, cut their cotyledons and hypocotyls, and place them respectively on callus induction media supplemented with 1 mg / L 2,4-D, 2 mg / L 2,4-D, 3 mg / L 2,4-D, 4 mg / L 2,4-D, and 5 mg / L 2,4-D in a medium of MS + 30 g / L sugar + 8 g / L agar, and conduct induction culture under dark conditions at 25 ± 2 °C.

[0037] The results showed that callus was induced in all experimental groups with a high callus induction rate (>90%), especially in the callus induction medium supplemented with 4 mg / L 2,4-D, where the callus induction rate was as high as 96% (as shown in Table 1).

[0038]

[0039] 2. Comparison of the callus induction rates with different concentrations of PSK

[0040] In a laminar flow hood, take the carnation seedlings cultured in step (2) of Example 1, cut their cotyledons and hypocotyls, and place them separately on the callus induction medium supplemented with 10 -5 mol / L and 10 -7 mol / L PSK in the medium of MS + 30 g / L sugar + 8 g / L agar + 4 mg / L 2,4-D, and conduct induction culture under dark conditions at 25 ± 2 °C.

[0041] The results showed that the callus induction medium supplemented with 10 -7 mol / L PSK could effectively induce the occurrence of callus, and the browning rate of the callus was relatively low (as shown in Table 2). Adding PSK helped the regeneration direction of carnation to follow the somatic embryogenesis pathway rather than the organogenesis pathway, facilitating the acquisition of embryogenic callus and highly efficient somatic embryo materials for carnation.

[0042]

[0043] 3. Using the callus induction medium of MS + 30 g / L sugar + 8 g / L agar + 4 mg / L 2,4-D + 10 -7 mol / L PSK, under dark conditions at 25 ± 2 °C, after inducing and culturing the cotyledons and hypocotyls of carnation for 45 days, callus with a compact texture and a golden yellow color was induced in the hypocotyls of carnation (as Figure 1 shown), and callus with a loose texture, golden yellow color, and fragility was induced in the cotyledons of carnation (as Figure 2 shown). Example 3

[0044] Transfer the cotyledon callus and hypocotyl callus of carnation obtained in Example 2 to the embryogenic callus induction medium respectively, with the culture conditions the same as in Example 1. After inducing and culturing for 40 days, embryogenic callus was obtained in the hypocotyl callus (as Figure 3 shown), and no embryogenic callus was produced in the cotyledon callus (as Figure 4 shown).

[0045] Transfer the embryogenic callus obtained from the hypocotyl to a differentiation medium of MS + 30 g / L sugar + 8 g / L agar + 1 mg / L NAA + 0.25 mg / L TDZ + 1 mg / L KT for differentiation culture. The culture conditions are the same as those in Example 1. Differentiate until obtaining Dianthus caryophyllus somatic embryos (as shown in Figure 5 , 6 ). Take the seedlings after differentiation of Dianthus caryophyllus somatic embryos and observe them under a stereomicroscope (as shown in Figure 7 ).

[0046] Respectively, put the Dianthus caryophyllus somatic embryo materials at different stages into 70% FAA fixative for fixation for 24 h, and then perform dehydration, infiltration with paraffin wax and embedding. After embedding, trim the wax blocks. Longitudinally cut the trimmed wax blocks into 4-μm sections with a paraffin microtome. After sectioning, dewax the materials, then stain with safranin for 1 h and with fast green for 1 min (the safranin-fast green paraffin section was entrusted to Wuhan Sevier Biotechnology Co., Ltd. to complete). Observe the stained paraffin sections with a biological microscope. The globular embryos are as shown in Figure 8 , and the heart-shaped embryos are as shown in Figure 9 . Example 4

[0047] The steps and culture conditions of this example are basically the same as those in Example 1, except that:

[0048] The basic medium is: MS + 30 g / L sugar + 8 g / L agar, pH = 6;

[0049] The callus induction medium is: MS + 30 g / L sugar + 8 g / L agar + 1 mg / L 2,4-D + 10 -6 mol / L PSK;

[0050] The embryogenic callus induction medium is: MS + 30 g / L sucrose + 8 g / L agar + 0.5 mg / L 6-BA + 2.0 mg / L NAA;

[0051] The differentiation medium is: MS + 30 g / L sugar + 8 g / L agar + 0.5 mg / L NAA + 0.1 mg / L TDZ + 2.5 mg / L KT;

[0052] The rooting medium is: MS + 30 g / L sugar + 8 g / L agar, pH = 6. Example 5

[0053] The steps and culture conditions of this example are basically the same as those in Example 1, except that:

[0054] The basic medium is: MS + 30 g / L sugar + 8 g / L agar, pH = 6.2;

[0055] The callus induction medium is: MS + 30 g / L sugar + 8 g / L agar + 3 mg / L 2,4-D + 10 -8 mol / L PSK;

[0056] The embryogenic callus induction medium is: MS + 30 g / L sugar + 8 g / L agar + 1 mg / L 6-BA + 1.5 mg / L NAA;

[0057] The differentiation medium is: MS + 30 g / L sugar + 8 g / L agar + 1.5 mg / L NAA + 1.0 mg / L TDZ + 1.5 mg / L KT;

[0058] The rooting medium is: MS + 30 g / L sugar + 8 g / L agar, pH = 6.2. Example 6

[0059] The steps and culture conditions of this example are basically the same as those of Example 1, except that:

[0060] The basal medium is: MS + 30 g / L sugar + 8 g / L agar, pH = 5.8;

[0061] The callus induction medium is: MS + 30 g / L sugar + 8 g / L agar + 5 mg / L 2,4-D + 10 -7 mol / L PSK;

[0062] The embryogenic callus induction medium is: MS + 30 g / L sugar + 8 g / L agar + 2.5 mg / L 6-BA + 0.1 mg / L NAA;

[0063] The differentiation medium is: MS + 30 g / L sugar + 8 g / L agar + 2.5 mg / L NAA + 2.0 mg / L TDZ + 0.5 mg / L KT;

[0064] The rooting medium is: MS + 30 g / L sugar + 8 g / L agar, pH = 5.8. Example 7

[0065] Using the method described in the present invention to regenerate carnation, the number of somatic embryo inductions per single embryogenic callus reaches 1,683, among which there are only 60 abnormal seedlings, and the abnormality rate is only 3.44% (as Figure 11 shown).

Claims

1. A method for constructing an efficient somatic embryo regeneration system of carnation, characterized in that, It includes the following steps: (1) Seed germination: The sterilized carnation seeds are inoculated onto the basic medium of MS + 30 g / L sugar + 8 g / L agar, pH = 6 ± 0.

2. First, they are cultured in the dark until the seeds germinate, and then they are cultured under light until carnation seedlings are obtained; (2) Callus induction: Take carnation seedlings, cut off the hypocotyls, and place them on a callus induction medium of MS + 30 g / L sugar + 8 g / L agar + 1 - 5 mg / L 2,4-D + 10 -6 - 10 -8 mol / L PSK, and culture them in the dark until callus is produced; (3) Embryogenic callus induction: The induced callus is transferred onto the embryogenic callus induction medium of MS + 30 g / L sugar + 8 g / L agar + 0.5 - 2.5 mg / L 6 - BA + 0.1 - 2.0 mg / L NAA, and cultured under light until embryogenic callus is produced; (4) Differentiation of embryogenic callus: The embryogenic callus is chopped and placed in the differentiation medium of MS + 30 g / L sugar + 8 g / L agar + 0.5 - 2.5 mg / L NAA + 0.1 - 2.0 mg / L TDZ + 0.5 - 2.5 mg / L KT, and cultured under light until somatic embryos differentiate into buds; (5) Rooting culture: The cotyledon embryos are separated from the embryogenic callus and inoculated into the medium of MS + 30 g / L sucrose + 8 g / L agar, pH = 6 ± 0.2, and cultured under light; after rooting, they are planted in a plug tray filled with substrate, and transplanted after adapting to the natural environment.

2. The method for constructing an efficient somatic embryo regeneration system of carnation according to claim 1, wherein The culture temperature in steps (1) to (4) is 25 ± 2 °C, and the culture temperature in step (5) is 23 ± 2 °C.

3. The construction method of the highly efficient somatic embryo regeneration system of carnation according to claim 1, characterized in that, In steps (1), (3), and (4), the photoperiod of the light incubation step is 16 h, and the light intensity is 36 μmol·m -2 ·s -1 ; in step (5), the photoperiod of the light incubation step is 16 h, and the light intensity is 40 μmol·m -2 ·s -1 .

4. The method for constructing an efficient somatic embryo regeneration system of carnation according to claim 1, characterized in that, The sterilization step in step (1) is: The carnation seeds are first soaked in 75% alcohol and then rinsed with sterile water, then soaked in 3% sodium hypochlorite solution and rinsed with sterile water, and then the moisture on the seed surface is removed.

5. The method for constructing an efficient somatic embryo regeneration system of carnation according to claim 1, characterized in that, The substrate in step (5) is composed of peat soil and perlite mixed in a mass ratio of 2:1.

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