Tissue culture method for seedlings of Jie apple

Abstract

The invention provides a tissue culture method for seedlings of apple trees, and belongs to the technical field of apple tissue culture breeding, the tissue culture method comprises the following steps: 1) disinfecting seeds of apple trees, removing seed coats, inoculating embryos after cotyledons are cut into a germination culture medium for germination, and obtaining tissue culture seedlings; and (2) cutting adventitious buds from the tissue culture seedlings, inoculating the adventitious buds into a proliferation culture medium, and carrying out proliferation culture to obtain seedlings. According to the tissue culture method, the embryos are used as explants, the embryos have better differentiation and propagation capacity, the regeneration capacity of the obtained tissue culture seedlings is higher than that of the tissue culture seedlings obtained by using other materials as explants, and the propagation coefficient is high.

Description

Technical Field

[0001] The invention belongs to the technical field of apple tissue culture and propagation, and in particular relates to a tissue culture method for maiden apple seedlings. Background Art

[0002] Apple variety resources are very rich, and there are many excellent varieties for fresh fruit processing and rootstock. The breeding cycle of apples is long, and gene editing breeding can modify specific genes, which has good application prospects in apple breeding. However, the genetic transformation of apples is very difficult, which makes it very difficult to apply gene editing technology in apple genetic improvement. One of the main constraints is that apple tissue culture seedlings are not easy to differentiate and have a low reproduction coefficient. At present, most scientific research institutes use the apple genetic transformation variety "Gala 3". "Gala 3" is an excellent seedling selected from "Gala" apples with a high reproduction coefficient and suitable for genetic transformation. In order to further expand the scope of application of gene editing in apple variety improvement, it is very necessary to provide more varieties with high reproduction coefficients and seedlings suitable for genetic transformation.

[0003] With the development of social economy and the rise of sightseeing fruit industry, Maiden Apple, as a small fruit variety of ornamental apple resources, has gradually attracted widespread attention due to its small size. And because its fruit can be fully colored, it is often used as a test material in fruit coloring experiments. However, the reproduction coefficient of apple tissue culture seedlings is affected by different factors, such as variety, explant type, type and concentration of plant hormones, etc. There is no method for cultivating seedlings with high reproduction coefficient and suitable for genetic transformation for Maiden Apple. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a tissue culture method for breeding high-reproduction-coefficient maiden apple seedlings using embryos as explants. The present invention has positive significance for genetic engineering genetic improvement, and also plays a vital role in the subsequent large-scale factory breeding of high-reproduction-coefficient high-quality seedlings and virus-free seedlings.

[0005] The present invention provides a tissue culture method for maiden apple seedlings, comprising the following steps:

[0006] 1) sterilizing the apple seeds, removing the seed coat, and inoculating the embryos after removing the cotyledons into a germination medium for germination to obtain tissue culture seedlings;

[0007] 2) cutting adventitious buds from the tissue culture seedlings and inoculating them into a proliferation medium for proliferation culture to obtain seedlings;

[0008] The proliferation culture medium is: MS+6-BA 0.5-0.80 mg / L+NAA 0-0.15 mg / L+IAA 0-0.15 mg / L+sucrose 25-35 g / L+agar powder 5-9 g / L, wherein NAA and IAA are not both 0.

[0009] Preferably, the proliferation culture medium is: MS+6-BA 0.7-0.75 mg / L+NAA 0.08-0.12 mg / L+sucrose 28-32 g / L+agar powder 6-7 g / L.

[0010] Preferably, the proliferation culture time is 25 to 35 days.

[0011] Preferably, in step 2), adventitious buds are cut from tissue culture seedlings with a seedling height of 1.5 to 2.0 cm, strong growth and similar growth potential.

[0012] Preferably, the germination medium is MS+6-BA 1.2-1.7 mg / L+NAA 0.1-0.3 mg / L+sucrose 45-55 g / L+agar powder 5-8 g / L.

[0013] Preferably, the germination comprises dark culture and normal light culture performed sequentially, the temperature of the dark culture is 3-5° C., and the time of the dark culture is 6-8 days.

[0014] Preferably, the temperature of the normal light culture is 25±2°C, the light cycle is 16h / 8h, the light intensity is 1500-2500lx, the air humidity is 70%-80%, and the time of the normal light culture is 28-32d.

[0015] Preferably, the disinfection described in step 1) comprises detergent cleaning, alcohol soaking and sodium hypochlorite solution soaking performed in sequence, the alcohol soaking time is 0.5 to 1.5 minutes, the sodium hypochlorite solution soaking time is 8 to 12 minutes, and the volume concentration of the sodium hypochlorite solution is 1.5 to 2.5%.

[0016] Preferably, it is characterized in that the maiden apple seeds in step 1) are mature, healthy and plump seeds.

[0017] Compared with the prior art, the present invention has the following beneficial effects: the tissue culture method of maiden apple seedlings provided by the present invention uses embryos as explants. Compared with traditional tissue culture explants, such as stem segments and adventitious buds, the embryos as explants have better differentiation and reproduction capabilities, and the obtained tissue culture seedlings have stronger regeneration capabilities and higher reproduction coefficients than tissue culture seedlings obtained with other materials as explants. The present invention further improves the reproduction coefficient by limiting the culture medium and culture conditions in the germination and proliferation culture stages; under the cultivation method of the present invention, maiden tissue culture seedlings have a higher reproduction coefficient, and the cultivated maiden tissue culture seedlings grow robustly and have firm leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure is a flow chart of the tissue culture method of the maiden apple seedlings of the present invention;

[0019] Figure 2 This is a diagram showing the growth status of the female tissue culture seedlings of the present invention in a proliferation medium containing growth regulators of different types and concentrations;

[0020] Figure 3 This is a diagram of female tissue culture seedlings with a high propagation coefficient cultivated using the tissue culture method selected by the present invention. DETAILED DESCRIPTION

[0021] The present invention provides a tissue culture method for maiden apple seedlings, comprising the following steps: 1) sterilizing maiden apple seeds, removing seed coats, and inoculating seed embryos after cutting off cotyledons into a germination medium for germination to obtain tissue culture seedlings; 2) cutting adventitious buds from the tissue culture seedlings, and inoculating them into a proliferation medium for proliferation culture to obtain seedlings.

[0022] In the present invention, first, the maiden apple seeds are sterilized and the seed coat is removed, and the embryos after the cotyledons are removed are inoculated into a germination medium for germination to obtain tissue culture seedlings. In the present invention, the maiden apple fruits are preferably harvested from mid-July to mid-August, and the seeds are peeled from the maiden apple fruits, and preferably mature and healthy and plump seeds are selected for subsequent operations. In the present invention, the maiden apple seeds are disinfected, and the disinfection preferably includes detergent cleaning, alcohol soaking and sodium hypochlorite solution soaking in sequence; in the specific implementation process of the present invention, it is preferred to use detergent to fully clean the seed surface, and then rinse the seeds with running water for 20 to 40 minutes; the rinsed seeds are preferably placed in a clean bench and soaked in alcohol, and the alcohol soaking time is preferably 0.5 to 1.5 minutes, more preferably 0.8 to 1.2 minutes, and more preferably 1 minute; the present invention soaks in sodium hypochlorite solution after the alcohol soaking, and the soaking time of the sodium hypochlorite solution is preferably 8 to 12 minutes, more preferably 9 to 11 minutes, and more preferably 10 minutes; the volume concentration of the sodium hypochlorite solution is preferably 1.5 to 2.5%, more preferably 1.8 to 2.2%, and more preferably 2.0%. After soaking in the sodium hypochlorite solution, the present invention preferably rinses with sterile water 3 to 5 times and transfers the seeds to a clean culture dish.

[0023] In the present invention, after the maiden apple seeds are sterilized, the seed coat is removed with a sterilized scalpel in a clean bench, the complete embryo is taken out with tweezers, and then the cotyledons of the embryo are removed with a scalpel. The embryo after the cotyledons are removed is inoculated in a germination medium for germination. In the present invention, the germination medium is preferably MS+6-BA 1.2-1.7 mg / L+NAA 0.1-0.3 mg / L+sucrose 45-55 g / L+agar powder 5-8 g / L, more preferably MS+6-BA 1.3-1.6 mg / L+NAA 0.15-0.25 mg / L+sucrose 48-52 g / L+agar powder 6-7 g / L, and even more preferably MS+6-BA 1.5 mg / L+NAA 0.2 mg / L+sucrose 50 g / L+agar powder 7 g / L. In the present invention, the germination includes dark culture and normal light culture carried out sequentially, the temperature of the dark culture is 3-5°C, more preferably 4°C, the time of the dark culture is preferably 6-8d, more preferably 7d; the temperature of the normal light culture is preferably 25±2°C, the light cycle is 16h / 8h, the light intensity is 1500-2500lx, the air humidity is 70%-80%, and the time of the normal light culture is preferably 28-32d, more preferably 29-31d, and even more preferably 30d.

[0024] After obtaining tissue culture seedlings, the present invention cuts adventitious buds from the tissue culture seedlings and inoculates them into a proliferation medium for proliferation culture to obtain seedlings. In the present invention, it is preferred to select tissue culture seedlings with a seedling height of 1.5 to 2.0 cm, strong growth, and similar growth potential to cut adventitious buds. In the present invention, the proliferation medium is preferably: MS+6-BA 0.5-0.80 mg / L+NAA 0-0.15 mg / L+IAA 0-0.15 mg / L+sucrose 25-35 g / L+agar powder 5-9 g / L, wherein NAA and IAA are not 0 at the same time; further preferably, it is MS+6-BA 0.7-0.75 mg / L+NAA 0.08-0.12 mg / L+sucrose 28-32 g / L+agar powder 6-7 g / L, and further preferably, it is MS+6-BA 0.75 mg / L+NAA 0.10 mg / L+sucrose 30 g / L+agar powder 7 g / L; or further preferably, it is MS+6-BA 0.70-0.80 mg / L+IAA 0.08-0.12 mg / L+sucrose 28-32 g / L+agar powder 6-8 g / L, more preferably MS+6-BA 0.75 mg / L+IAA 0.10 mg / L+sucrose 30 g / L+agar powder 7 g / L. In the present invention, the time of the proliferation culture is preferably 25-35 days, more preferably 28-32 days, and more preferably 30 days. The present invention obtains seedlings after proliferation culture, which grow robustly and have firm leaves.

[0025] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0026] Example

[0027] Determination of the composition of the germination medium

[0028] Step S1, fruit harvesting: harvesting maidenhair fruits from mid-July to mid-August;

[0029] Step S2, seed collection: peel the seeds from the maiden apple fruit, and select mature, healthy and plump seeds for use.

[0030] Step S3, aseptic tissue culture seedling acquisition: take fruit seeds, clean the seed surface with detergent, rinse with running water for 0.5h, transfer to a clean bench, soak and disinfect with 75% (volume fraction) alcohol for 1min, then soak and disinfect with 2% (volume fraction) sodium hypochlorite solution for 10min, rinse with sterile water 5 times, transfer to a clean culture dish, use a sterilized scalpel to remove the seed coat, take out the complete embryo with tweezers, and then use a scalpel to cut off the cotyledon of the embryo. The embryos after the cotyledons are removed are inoculated into germination mediums of different compositions, placed in a dark culture medium at 4°C for 7d, and then transferred to normal light conditions, with a temperature of 25±2°C, a photoperiod of 16h / 8h, a light intensity of 1500-2500lx, and an air humidity of 70%-80%, cultured for 30d, and the embryo germination results are counted.

[0031] Among them, the germination medium was screened with MS as the basic medium and L9(3 3 ) Orthogonal test method, select different concentrations and ratios of 6-BA, NAA, GA3 hormone combination culture medium, 6-BA concentration set three concentration gradients, respectively 0.5mg / L, 1.0mg / L and 1.5mg / L; NAA concentration set three concentration gradients, respectively 0.05mg / L, 0.1mg / L and 0.2mg / L; GA3 concentration set three concentration gradients, respectively 0mg / L, 1.0mg / L and 2.0mg / L. Sucrose 50g / L, agar powder 7g / L were added to each culture medium, and the pH value was adjusted to 5.8-6.0.

[0032] There were 9 treatments in this experiment, and each treatment was repeated for 3 groups. Each bottle of culture medium was inoculated with 4 to 5 seeds, and each culture medium was inoculated with 30 seeds. The statistical results of embryo germination are shown in Table 1.

[0033] Table 1 Germination rate of embryos in different germination media

[0034]

[0035]

[0036] Note: T value indicates the mean value of the same level in the same column. The same letters in the same column indicate that there is no significant difference at the 0.05 level after Duncan's new multiple range test.

[0037] The data analysis showed that the order of priority of the three factors in the orthogonal test was 6-BA, GA3, and NAA, and the differences between the three factor levels were significant. Further test results showed that when the concentration of 6-BA was 1.5 mg / L and the concentration of NAA was 0.2 mg / L, the germination rate of the embryo after the cotyledon was removed was the highest, reaching 76.67%. Therefore, the germination medium formula used in the present invention is MS+6-BA1.5 mg / L+NAA 0.2 mg / L+sucrose 50 g / L+agar powder 7 g / L.

[0038] Proliferation medium composition determination

[0039] Process 1

[0040] Step S1, fruit harvesting: harvesting maidenhair fruits from mid-July to mid-August;

[0041] Step S2, seed collection: peel the seeds from the maiden apple fruit, and select mature, healthy and plump seeds for use.

[0042] Step S3, obtaining sterile tissue culture seedlings: take fruit seeds, clean the seed surface thoroughly with detergent, rinse with running water for 0.5h, transfer to an ultra-clean workbench, soak and disinfect with 75% (volume fraction) alcohol for 1min, then soak and disinfect with 2% (volume fraction) sodium hypochlorite solution for 10min, rinse with sterile water 5 times, transfer to a clean culture dish, use a sterilized scalpel to open the seed coat, use tweezers to take out the complete embryo, and then use a scalpel to remove the cotyledons of the embryo. The embryos after the cotyledons were removed were inoculated into the screened germination medium (MS+6-BA 1.5mg / L+NAA 0.2mg / L+sucrose 50g / L+agar powder 7g / L), 4 to 5 seeds were inoculated into each bottle of culture medium, and placed in a dark culture at 4°C for 7 days, and then transferred to normal light conditions with a temperature of 25±2°C, a light cycle of 16h / 8h, a light intensity of 1500-2500lx, and an air humidity of 70%-80%. After culture for 30 days, the embryos germinated into seedlings for later use.

[0043] Step S4, proliferation culture: select tissue culture seedlings with a seedling height of 1.5 cm to 2.0 cm, strong growth, and the same or similar growth potential, cut adventitious buds, and inoculate them into a proliferation culture medium. Under normal lighting conditions, the temperature is 25±2°C, the light cycle is 16h / 8h, the light intensity is 1500~2500lx, and the air humidity is 70%~80%. After culturing for 30 days, the proliferation coefficient of the adventitious buds is counted.

[0044] The composition of P1 proliferation medium is as follows: MS+6-BA 0.5 mg / L+sucrose 30 g / L+agar powder 7 g / L.

[0045] Process 2

[0046] The steps are the same as those described in Treatment 1 above, except that the composition of the proliferation medium is different;

[0047] The composition of P2 proliferation medium is as follows: MS+6-BA 0.5mg / L+NAA 0.3mg / L+IAA 0.1mg / L+sucrose 30g / L+agar powder 7g / L.

[0048] Process 3

[0049] The steps are the same as those described in Treatment 1 above, except that the composition of the proliferation medium is different;

[0050] The composition of P3 proliferation medium is as follows: MS+6-BA 0.5 mg / L+NAA 0.1 mg / L+IAA 0.5 mg / L+sucrose 30 g / L+agar powder 7 g / L.

[0051] Process 4

[0052] The steps are the same as those described in Treatment 1 above, except that the composition of the proliferation medium is different;

[0053] The composition of P4 proliferation medium is as follows: MS+6-BA 0.75 mg / L+NAA 0.3 mg / L+IAA 0.5 mg / L+sucrose 30 g / L+agar powder 7 g / L.

[0054] Process 5

[0055] The steps are the same as those described in Treatment 1 above, except that the composition of the proliferation medium is different;

[0056] The composition of P5 proliferation medium is as follows: MS+6-BA 0.75 mg / L+IAA 0.1 mg / L+sucrose 30 g / L+agar powder 7 g / L.

[0057] Process 6

[0058] The steps are the same as those described in Treatment 1 above, except that the composition of the proliferation medium is different;

[0059] The composition of P6 proliferation medium is as follows: MS+6-BA 0.75 mg / L+NAA 0.1 mg / L+sucrose 30 g / L+agar powder 7 g / L.

[0060] Process 7

[0061] The steps are the same as those described in Treatment 1 above, except that the composition of the proliferation medium is different;

[0062] The composition of P7 proliferation medium is as follows: MS+6-BA 1.0 mg / L+NAA 0.1 mg / L+IAA 0.1 mg / L+sucrose 30 g / L+agar powder 7 g / L.

[0063] Process 8

[0064] The steps are the same as those described in Treatment 1 above, except that the composition of the proliferation medium is different;

[0065] The composition of P8 proliferation medium is as follows: MS+6-BA 1.0 mg / L+NAA 0.3 mg / L+sucrose 30 g / L+agar powder 7 g / L.

[0066] Process 9

[0067] The steps are the same as those described in Treatment 1 above, except that the composition of the proliferation medium is different;

[0068] The composition of P9 proliferation medium is as follows: MS+6-BA 1.0 mg / L+IAA 0.5 mg / L+sucrose 30 g / L+agar powder 7 g / L.

[0069] Finally, tissue culture seedlings with high propagation coefficients were obtained, and the proliferation coefficients of adventitious buds in proliferation medium containing different types and concentrations of growth regulators were compared. The obtained data are shown in Table 2.

[0070] Table 2 Proliferation coefficient and growth status of tissue culture seedlings in different proliferation media

[0071]

[0072]

[0073] By analyzing the data in Table 2, it can be found that in P6 medium, that is, when the concentration of 6-BA is 0.75 mg / L and the concentration of NAA is 0.1 mg / L, the proliferation coefficient of the female tissue culture seedlings is the highest, reaching 7.69; in P3 medium, that is, when the concentration of 6-BA is 0.5 mg / L, the concentration of NAA is 0.1 mg / L, and the concentration of IAA is 0.5 mg / L, the proliferation coefficient of the female tissue culture seedlings is the lowest, which is 2.40. Figure 2 It can be seen that the number of plants proliferating in P5 medium (6-BA concentration of 0.75 mg / L, IAA concentration of 0.1 mg / L) is greater, and the growth state of plants in P6 medium (6-BA concentration of 0.75 mg / L, NAA concentration of 0.1 mg / L) is better, and the leaves are stretched and firm. Finally, the P6 medium, i.e., the medium formula is determined to be MS+6-BA 0.75 mg / L+NAA 0.1 mg / L+sucrose 30 g / L+agar powder 7 g / L, which is the optimal proliferation medium formula for high proliferation coefficient of female tissue culture seedlings.

[0074] The existing technology "Research on the proliferation and rooting culture of apple rootstock G.11 test tube seedlings" (Guo Jing, Chai Cijiang, Shi Yanshan, Luo Jianxia, ​​Jiang Wen. Research on the proliferation and rooting culture of apple rootstock G.11 test tube seedlings [J]. Journal of Tianjin Agricultural College, 2019, 26(04): 38-42+48.) found that the reproduction coefficient of G.11 test tube seedlings was the highest in MS medium containing 6-BA 0.5 mg / L, which can reach 6.70; the existing technology "Research on tissue culture and callus induction system of apple dwarfing rootstock 'T337'" (Feng Mingjun, Lei Fuchen, Dou Jing, Wang Xinjian. Research on tissue culture and callus induction system of apple dwarfing rootstock 'T337' [J]. Journal of Tarim University, 2021, 33(04): 45-52.) took the best explant of 'T337'-dormant buds as the experimental material, and found that the proliferation culture was in 6-BA 0.75 mg / L+IBA The maximum proliferation coefficient under the hormone combination of 0.50 mg / L is 2.60. The tissue culture method provided by the present invention, by limiting the composition of the germination medium and the proliferation medium and the specific method steps, obtains a maximum proliferation coefficient of 7.69 for the female tissue culture seedlings, which has achieved unexpected technical effects compared with the prior art.

[0075] It can be seen from the above embodiments that the tissue culture seedlings obtained by the tissue culture method of maiden apple seedlings provided by the present invention have strong regeneration ability and high reproduction coefficient; the cultivated maiden tissue culture seedlings grow robustly and have firm leaves.

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for tissue culture of maiden apple seedlings, characterized in that: The following steps are involved: 1) sterilizing the apple seeds, removing the seed coat, and inoculating the embryos after removing the cotyledons into a germination medium for germination to obtain tissue culture seedlings; 2) cutting adventitious buds from the tissue culture seedlings and inoculating them into a proliferation medium for proliferation culture to obtain seedlings; The proliferation culture medium is: MS+6-BA 0.5-0.80 mg / L+NAA 0-0.15 mg / L+IAA 0-0.15 mg / L+sucrose 25-35 g / L+agar powder 5-9 g / L, wherein NAA and IAA are not both 0.

2. The tissue culture method according to claim 1, characterized in that The proliferation culture medium is: MS+6-BA 0.7-0.75 mg / L+NAA 0.08-0.12 mg / L+sucrose 28-32 g / L+agar powder 6-7 g / L.

3. The tissue culture method according to claim 1 or 2, characterized in that: The proliferation culture time is 25 to 35 days.

4. The tissue culture method according to claim 3, characterized in that: Step 2) Select tissue culture seedlings with a seedling height of 1.5 to 2.0 cm, strong growth and similar growth potential to cut adventitious buds.

5. The tissue culture method according to claim 1, characterized in that: The germination medium is MS+6-BA 1.2-1.7 mg / L+NAA 0.1-0.3 mg / L+sucrose 45-55 g / L+agar powder 5-8 g / L.

6. The tissue culture method according to claim 5, characterized in that: The germination includes dark culture and normal light culture performed sequentially, the temperature of the dark culture is 3-5° C., and the time of the dark culture is 6-8 days.

7. The tissue culture method according to claim 6, characterized in that: The temperature of the normal light culture is 25±2° C., the light cycle is 16h / 8h, the light intensity is 1500-2500lx, the air humidity is 70%-80%, and the time of the normal light culture is 28-32d.

8. The tissue culture method according to claim 1, characterized in that: The disinfection described in step 1) includes detergent cleaning, alcohol soaking and sodium hypochlorite solution soaking in sequence, the alcohol soaking time is 0.5 to 1.5 minutes, the sodium hypochlorite solution soaking time is 8 to 12 minutes, and the volume concentration of the sodium hypochlorite solution is 1.5 to 2.5%.

9. The tissue culture method according to claim 1, characterized in that: The maiden apple seeds described in step 1) are mature, healthy and full seeds.

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