A method for promoting the germination of Quercus virginiana somatic embryos by alternating drying and rehydration culture

Abstract

The present invention provides a method for promoting the germination of Quercus virginiana somatic embryos by alternating drying and rehydration culture, belonging to the technical field of plant tissue culture. The method comprises the following steps: (1) taking immature zygotic embryos of Quercus virginiana as explants for somatic embryo induction to obtain embryogenic callus; (2) transferring the embryogenic callus obtained in step (1) into a proliferation medium for dark culture to obtain cotyledon embryos; (3) performing an alternating drying and rehydration treatment on the cotyledon embryos; (4) performing somatic embryo germination culture on the cotyledon embryos that have been subjected to the alternating drying and rehydration treatment in step (3) to obtain test-tube seedlings of Quercus virginiana. After inducing and proliferating the immature zygotic embryos of Quercus virginiana, the present invention performs alternating drying and rehydration culture, which can shorten the germination time of Quercus virginiana somatic embryos and improve the germination rate.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for promoting embryo germination of Virginia oak by alternate desiccation-rehydration culture. Background Art

[0002] Virginia oak ( Quercus virginiana ) belongs to Fagaceae ( Fagaceae )Quercus Quercus ) tree species, native to the United States, is an evergreen broad-leaved tall tree and an important coastal shelterbelt tree species on the southeastern coast of the United States. Virginia oak can tolerate high concentrations of salt spray and high soil salinity. In addition, Virginia oak also has a strong tolerance to heavy metals. It has been widely used in coastal afforestation, plain greening and mine vegetation restoration in the southeastern coast of my country. It is an important ecological tree species. At the same time, Virginia oak wood (oak) has high density, good material quality and high economic value; its fruit is rich in starch, which can provide a food source for birds and animals, and is of great significance to enriching biodiversity. Therefore, Virginia oak has a great demand in the Yangtze River Delta market due to its good stress resistance, economic value and multiple uses.

[0003] At present, most of the seedlings of Virginia oak are propagated by seeds. However, due to the large variation of seedlings, the uniformity of seedlings is poor, which affects its market value. Although the asexual propagation technology of Virginia oak has been solved by cutting and tissue culture methods in the early stage, the early tissue culture is a tissue culture seedling regeneration system based on stem segments with buds, which only solves the asexual propagation of Virginia oak. With the demand for genetic improvement of Virginia oak, the construction of Virginia oak genetic transformation system is also imminent, but there is no mature transformation system at present. In this field, the somatic embryogenesis and test tube seedling regeneration technology based on embryonic callus are the basis for plant genetic transformation. Although immature zygotic embryos can induce the production of embryonic callus, the germination rate of somatic embryos has been low, and some somatic embryos do not even germinate, which seriously restricts the construction of Virginia oak somatic embryo regeneration pathway.

[0004] Somatic embryo drying treatment is used to promote the germination of somatic embryos of many conifers. For example, proper drying pretreatment before somatic embryo germination can improve the germination rate of somatic embryos of spruce, Pinus sylvestris, etc. However, due to many factors affecting the drying treatment, the effects vary due to different drying treatment methods, drying time and tree species.

[0005] Based on the above problems, we hope to find a drying method that can specifically promote the germination of somatic embryos of Virginia oak, increase the germination rate of somatic embryos, and facilitate the construction of the somatic embryo regeneration pathway of Virginia oak. Summary of the invention

[0006] The object of the present invention is to provide a treatment method for promoting the germination of Quercus virginiana somatic embryos and improving the somatic embryo germination rate.

[0007] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0008] A method for promoting the germination of Quercus virginiana somatic embryos by alternate drying and rehydration culture, comprising the following steps:

[0009] (1) Taking immature zygotic embryos of Quercus virginiana as explants for somatic embryo induction to obtain embryogenic callus;

[0010] (2) Transferring the embryogenic callus obtained in step (1) into a proliferation medium for dark culture to obtain cotyledon embryos;

[0011] (3) Subjecting the cotyledon embryos obtained in step (2) to alternate drying and rehydration culture;

[0012] (4) Transferring the cotyledon embryos treated by alternate drying and rehydration in step (3) into an MS medium rich in amino acids for somatic embryo germination culture to obtain Quercus virginiana test-tube seedlings.

[0013] Preferably, the collection time of the explants in step (1) is from mid-July to late August of each year.

[0014] Preferably, the somatic embryo induction in step (1) is: placing the immature zygotic embryos in an induction medium, and under the conditions of pH 5.6 - 5.8 and 23 - 25 °C, performing dark culture for 28 - 31 days.

[0015] Preferably, the composition of the induction medium is: MS + casein hydrolysate 200 - 500 mg / L + glutamine 0.1 - 1 g / L + proline 0.5 - 5 mmol / L + 0.044 - 0.44 μM 6 - BA.

[0016] Preferably, the composition of the proliferation medium in step (2) is: MS + casein hydrolysate 200 - 500 mg / L + glutamine 0.1 - 1 g / L + proline 0.5 - 5 mmol / L.

[0017] Preferably, the alternate drying and rehydration culture in step (3) is: drying the cotyledon embryos for 3 - 7 d, then transferring them into a 1 / 4MS low - salt medium for rehydration for 3 - 7 d. The culture environments for both drying and rehydration are dark culture, and the rehydrated cotyledon embryos are dried again for 2 - 4 d in a light environment.

[0018] Preferably, the drying treatment is natural drying using a double - layer petri dish, drying treatment using filter paper, or drying treatment using a chemical reagent; the chemical reagent is a saturated Ca(NO3)2·4H2O solution.

[0019] Preferably, the composition of the amino acid-rich MS medium is: MS + casein hydrolysate 200-500 mg / L + glutamine 0.1-1 g / L + proline 0.5-5 mmol / L.

[0020] Preferably, the 1 / 4MS low-salt medium is obtained by reducing the content of macroelements in the amino acid-rich MS medium to 1 / 4, while keeping the contents of other trace elements, organic substances, iron salts, sucrose, plant gel and amino acids unchanged.

[0021] Preferably, the culture conditions for somatic embryo germination are: temperature 23-25 °C, light-dark cycle 16h / 8h, light intensity 250-300 µmol·m -2 ·s -1 . Beneficial effects

[0022] After inducing and proliferating the immature zygotic embryos of Quercus virginiana by the method of alternating drying and rehydration culture provided by the present invention, after drying treatment, they are transferred to a hormone-free 1 / 4MS medium for rehydration, and then dried again, which can shorten the germination time of somatic embryos of Quercus virginiana and improve the germination rate.

[0023] The test results show that the germination rate of the alternating drying and rehydration culture method of the present invention has been improved to varying degrees, and the germination time can also be significantly shortened. Among them, the germination time of the chemical reagent drying-rehydration treatment is shortened by 36% compared with that of the chemical reagent drying treatment, and the germination rate is increased by 7.3%. Description of the drawings

[0024] Figure 1 It is the test-tube seedling of Quercus virginiana obtained after somatic embryo germination in Example 1.

[0025] Figure 2 It is the state diagram of somatic embryos after induction culture of the explants obtained on July 12 in Test Example 1.

[0026] Figure 3 It is the state diagram of somatic embryos after induction culture of the explants obtained on July 26 in Test Example 1.

[0027] Figure 4 It is the state diagram of somatic embryos after induction culture of the explants obtained on August 9 in Test Example 1.

[0028] Figure 5 It is the state diagram of somatic embryos after induction culture of the explants obtained on August 25 in Test Example 1.

[0029] Figure 6Figure of embryogenic callus induced by the culture medium of Formula 1 in Test Example 2.

[0030] Figure 7 Figure of cell morphology of embryogenic callus induced by the culture medium of Formula 1 in Test Example 2.

[0031] Figure 8 Figure of embryogenic callus induced by the culture medium of Formula 2 in Test Example 2.

[0032] Figure 9 Figure of cell morphology of embryogenic callus induced by the culture medium of Formula 2 in Test Example 2.

[0033] Figure 10 Figure of embryogenic callus induced by the culture medium of Formula 3 in Test Example 2.

[0034] Figure 11 Figure of cell morphology of embryogenic callus induced by the culture medium of Formula 3 in Test Example 2.

[0035] Figure 12 Figure of embryogenic callus induced by the culture medium of Formula 4 in Test Example 2.

[0036] Figure 13 Figure of cell morphology of embryogenic callus induced by the culture medium of Formula 4 in Test Example 2.

[0037] Figure 14 Figure of embryogenic callus induced by the culture medium of Formula 5 in Test Example 2.

[0038] Figure 15 Figure of cell morphology of embryogenic callus induced by the culture medium of Formula 5 in Test Example 2.

[0039] Figure 16 Figure of embryogenic callus induced by the culture medium of Formula 6 in Test Example 2.

[0040] Figure 17 Figure of cell morphology of embryogenic callus induced by the culture medium of Formula 6 in Test Example 2.

[0041] Figure 18 Figure of embryogenic callus induced by the culture medium of Formula 7 in Test Example 2.

[0042] Figure 19 Figure of cell morphology of embryogenic callus induced by the culture medium of Formula 7 in Test Example 2.

[0043] Figure 20 Schematic diagram of treatment by chemical reagent drying method.

[0044] Figure 21 Schematic diagram of treatment by double-layer petri dish drying method.

[0045] Figure 22 It is a schematic diagram of the filter paper drying method. Detailed implementation mode

[0046] In the present invention, the gelling agent in the MS medium is 0.24% plant gel.

[0047] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Example 1

[0048] A method for promoting the germination of Quercus virginiana somatic embryos by alternating drying and rehydration culture, comprising the following steps:

[0049] (1) Collect 200 immature seeds of Quercus virginiana in mid-July, rinse them thoroughly with dishwashing liquid, then rinse them 3 times with sterile water, then disinfect them with 70% alcohol for 1 minute and rinse them 3 times with sterile water; then disinfect them with mercuric chloride for 8 minutes, add 1 drop of Tween, and rinse them 3 times with sterile water;

[0050] Remove the immature zygotic embryos at the top of the disinfected seeds on a sterile workbench, inoculate them into the induction medium, and perform dark culture at 25°C for 30 days to obtain embryogenic callus;

[0051] The composition of the induction medium is MS + 0.44 μM 6-BA + casein hydrolysate 500 mg / L + glutamine 1 g / L + proline 5 mmol / L, pH 5.7.

[0052] (2) Transfer the embryogenic callus obtained in step (1) into the proliferation medium for dark culture;

[0053] The composition of the proliferation medium is: MS + casein hydrolysate 500 mg / L + glutamine 1 g / L + proline 5 mmol / L.

[0054] (3) Perform alternating drying and rehydration culture on the tissue cultured to cotyledon embryos:

[0055] Place the cotyledon embryos in an empty petri dish with a diameter of 9 cm, then place the petri dish in a large petri dish with a diameter of 12 cm, add 10 ml of sterilized saturated Ca(NO3)2·4H2O solution to the large petri dish, perform drying treatment at 25°C for 5 days, then transfer them to 1 / 4MS low-salt medium for dark culture and rehydration for 5 days, and perform the above treatment in the drying device again for 3 days for the rehydrated cotyledon embryos, and change the culture environment to light culture, with a light-dark cycle of 16 h / 8 h and a light intensity of 300 µmol·m -2 ·s -1 .

[0056] (4) Transfer the cotyledon embryos that have been subjected to the drying-rehydration alternation treatment in step (3) into the MS medium rich in amino acids, and carry out somatic embryo germination culture under the conditions of a temperature of 25 °C, a light-dark cycle of 16 h / 8 h, and a light intensity of 300 µmol·m -2 ·s -1 to obtain test-tube seedlings of Quercus virginiana ( Figure 1 ).

[0057] The composition of the MS medium rich in amino acids is: MS + 1 mg / L 6-BA + 500 mg / L casein hydrolysate + 1 g / L glutamine + 5 mmol / L proline;

[0058] The 1 / 4MS low-salt medium reduces the content of macronutrients in the MS medium rich in amino acids to 1 / 4, while the contents of other trace elements, organic substances, iron salts, sucrose, plant gel, and amino acids remain unchanged. Example 2

[0059] Different from Example 1, when performing the drying treatment in step (3), no chemical reagent was placed. Instead, the somatic embryos of Quercus virginiana were placed in an empty sterile petri dish, then the petri dish was placed in another larger petri dish, and finally the larger petri dish was sealed. Example 3

[0060] Different from Example 1, when performing the drying treatment in step (3), no chemical reagent was placed. Instead, the somatic embryos of Quercus virginiana were placed in a sterile petri dish lined with double-layer filter paper, the petri dish was placed in another larger petri dish, sterile water was added to the larger petri dish, and finally the larger petri dish was sealed. Comparative Example 1

[0061] Different from Example 1, the somatic embryos after the drying treatment in step (3) were directly transferred to the MS medium rich in amino acids described in Example 1 for germination test without performing the rehydration treatment. Comparative Example 2

[0062] Different from Example 2, the somatic embryos after the drying treatment in step (3) were directly transferred to the MS medium rich in amino acids described in Example 1 for germination test without performing the rehydration treatment. Comparative Example 3

[0063] Different from Example 3, the somatic embryos after the drying treatment in step (3) were directly transferred to the MS medium rich in amino acids described in Example 1 for germination test without performing the rehydration treatment.

[0064] Test Example 1 Somatic Embryo Status of Explants at Different Acquisition Times

[0065] Explants were collected and obtained on July 12, July 26, August 9, and August 25 respectively, and induced culture was carried out. The somatic embryo states of the explants obtained at different times were observed and marked after sampling.

[0066] The results showed that: after culturing the explants obtained at different times, the somatic embryo states were different. The explants from mid-July to late August and mid-August (such as Figure 2 , 3 , as shown in 4) had the best somatic embryo states, showing milky white, transparent, soft texture, while the explants from late August to September and later (such as Figure 5 shown) had callus with a darker color and a more brittle texture.

[0067] Test Example 2 Influence of Embryogenic Callus Induced by Different Medium Formulations

[0068] Different from Example 1, the formulations of the induction medium used in step (1) of this test example were respectively:

[0069] Formulation 1: MS + casein hydrolysate 500 mg / L + glutamine 1 g / L + proline 5 mmol / L + 0.45 μM 2,4-D (callus morphology see Figure 6 , cell morphology see Figure 7 );

[0070] Formulation 2: MS + casein hydrolysate 500 mg / L + glutamine 1 g / L + proline 5 mmol / L + 2.3 μM 2,4-D (callus morphology see Figure 8 , cell morphology see Figure 9 );

[0071] Formulation 3: MS + casein hydrolysate 500 mg / L + glutamine 1 g / L + proline 5 mmol / L + 4.5 μM 2,4-D (callus morphology see Figure 10 , cell morphology see Figure 11 );

[0072] Formulation 4: MS + casein hydrolysate 500 mg / L + glutamine 1 g / L + proline 5 mmol / L + 0.44 μM 6-BA (callus morphology see Figure 12 , cell morphology see Figure 13 );

[0073] Formulation 5: MS + casein hydrolysate 500 mg / L + glutamine 1 g / L + proline 5 mmol / L + 0.44 μM 6-BA + 0.45 μM 2,4-D (callus morphology see Figure 14 , cell morphology see Figure 15 );

[0074] Formula 6: MS + casein hydrolysate 500 mg / L + glutamine 1 g / L + proline 5 mmol / L + 0.44 μM 6-BA + 2.3 μM 2,4-D (see the callus morphology in Figure 16 , and the cell morphology in Figure 17 );

[0075] Formula 7: MS + casein hydrolysate 500 mg / L + glutamine 1 g / L + proline 5 mmol / L + 0.44 μM 6-BA + 4.5 μM 2,4-D (see the callus morphology in Figure 18 , and the cell morphology in Figure 19 );

[0076] Observing the morphology and cell conditions of embryogenic callus with different medium formulas, it can be seen that the callus and cell morphologies obtained with different medium formulas are different. Among them, the somatic embryo cells in the group of MS + casein hydrolysate 500 mg / L + glutamine 1 g / L + proline 5 mmol / L + 0.44 μM 6-BA are the most obvious.

[0077] Test Example 3 Effects of Different Drying Treatments on Somatic Embryo Germination Time and Germination Rate

[0078] Statistics on the effects of different drying treatments in Examples 1-3 and Comparative Examples 1-3 on somatic embryo germination time and germination rate are shown in Table 1: (The three drying treatment methods are respectively named the chemical reagent method (calcium nitrate method) (such as Figure 20 ), the double-layer petri dish drying method (such as Figure 21 ), and the filter paper drying method (such as Figure 22 ))

[0079] Table 1 Effects of Different Treatment Methods on Somatic Embryo Germination Rate

[0080]

[0081] As can be seen from Table 1, compared with only one drying treatment, the germination rate of the drying-rehydration alternating culture method has been improved to varying degrees, and the germination time can also be significantly shortened. Among them, the germination time of the chemical reagent drying-rehydration treatment is shortened by 36% compared with the chemical reagent drying treatment, and the germination rate is increased by 7.3%, with the most obvious effect.

[0082] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for promoting the germination of Quercus virginiana somatic embryos by alternating drying and rehydration culture, characterized in that, It includes the following steps: (1) Taking the immature zygotic embryos of Quercus virginiana as explants for somatic embryo induction to obtain embryogenic callus; the collection time of the explants is from mid-July to late August every year; (2) Transferring the embryogenic callus obtained in step (1) into a proliferation medium for dark culture to obtain cotyledon embryos; (3) Performing alternate drying-rehydration culture on the cotyledon embryos obtained in step (2); (4) Transferring the cotyledon embryos treated by alternate drying-rehydration in step (3) into an MS medium rich in amino acids for somatic embryo germination culture to obtain test-tube seedlings of Quercus virginiana; The alternate drying-rehydration culture is as follows: drying the cotyledon embryos for 3 - 7 days, then transferring them into a 1 / 4MS low-salt medium for rehydration for 3 - 7 days. The culture environments for both drying and rehydration are dark culture. After rehydration, the cotyledon embryos are dried again for 2 - 4 days in a light environment; The drying treatment is to place the cotyledon embryos in a small petri dish of a double-layer petri dish, and then add a chemical reagent into the large petri dish for drying treatment; The chemical reagent is a saturated Ca(NO3)2·4H2O solution; The composition of the induction medium is: MS + casein hydrolysate 200 - 500 mg / L + glutamine 0.1 - 1 g / L + proline 0.5 - 5 mmol / L + 0.044 - 0.44 μM 6-BA; The composition of the proliferation medium in step (2) is: MS + casein hydrolysate 200 - 500 mg / L + glutamine 0.1 - 1 g / L + proline 0.5 - 5 mmol / L; The composition of the MS medium rich in amino acids is: MS + casein hydrolysate 200 - 500 mg / L + glutamine 0.1 - 1 g / L + proline 0.5 - 5 mmol / L; The 1 / 4MS low-salt medium is to reduce the content of macroelements in the MS medium rich in amino acids to 1 / 4, while the contents of other trace elements, organic substances, iron salts, sucrose, plant gel, and amino acids remain unchanged.

2. The method according to claim 1, wherein the somatic embryo induction in step (1) is: placing the immature zygotic embryos in an induction medium, and performing dark culture for 28 - 31 days under the conditions of pH 5.6 - 5.8 and 23 - 25°C.

3. The method according to claim 2, wherein The culture conditions for the germination of somatic embryos are as follows: temperature 23 - 25°C, light - dark cycle 16h / 8h, light intensity 250 - 300 µmol·m -2 ·s -1 .

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