A method for establishing a bud regeneration system using peach immature embryos

Abstract

The invention discloses a method for establishing a bud regeneration system by using peach immature embryos. The method comprises the following steps: (1) taking peach immature embryos as explants, inoculating the immature embryos into an immature embryo induction callus medium for culturing to obtain callus tissue; the immature embryo induction callus medium is an MS medium containing 0.25-0.5 mg / L 6-BA, 0.05-0.25 mg / L TDZ, 0.2-0.5 mg / L 2,4-D, and 0.1-0.2 mg / L NAA, and having a pH value of 5.6-5.8; (2) cutting cotyledons from the callus tissue along the base of the embryo, inoculating the cotyledons into an adventitious bud induction medium for culturing to obtain adventitious bud clusters; the cotyledon induction adventitious bud medium is an MS medium containing 1-3 mg / L 6-BA, 0.1 mg / L IBA, and 100 mg / L inositol, and having a pH value of 5.6-5.8; and (3) transferring the adventitious bud clusters into a regeneration bud induction medium for culturing to obtain regenerated buds. This provides the possibility for future research on the genetic function of peaches and rapid improvement of fruit trees and fruit quality, and has important economic value and social benefits.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, and in particular to a method for establishing a bud regeneration system by utilizing peach immature embryos. Background Art

[0002] Peach (Prunus persica L.), originating in the Shaanxi-Gansu region of my country, is an important cash crop. In recent years, fruit quality has declined with increasing cultivation years and the development of new varieties that are more resistant to storage and transportation. While the application of genetic engineering breeding techniques has greatly advanced the breeding of Rosaceae fruit trees such as apple and plum, peach breeding has been slow due to difficulties in regeneration. Transgenic breeding and molecular design breeding techniques can break down species boundaries, significantly improving breeding efficiency and demonstrating significant potential in increasing yield, improving quality, and enhancing stress resistance.

[0003] Both technologies are based on genetic transformation. However, the current regeneration and genetic transformation efficiencies of peach materials are extremely low, preventing widespread application in production. This severely limits genetic transformation and functional research in peaches, hindering crop biotechnology improvement. Although many researchers have made progress in peach genotypes, regeneration systems, and genetic transformation systems in recent years, very few have been found to be able to regenerate and stably undergo genetic transformation. Therefore, establishing a peach regeneration system is crucial for improving the efficiency of peach genetic transformation. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of the present invention is to provide a method for establishing a bud regeneration system using peach embryos.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a method for establishing a bud regeneration system using peach immature embryos, comprising the following steps:

[0007] (1) taking peach immature embryos as explants, inoculating them into an immature embryo callus induction medium for culture, and obtaining callus tissue; the immature embryo callus induction medium is: MS medium containing 0.25-0.5 mg / L 6-BA, 0.05-0.25 mg / L TDZ, 0.2-0.5 mg / L 2,4-D, and 0.1-0.2 mg / L NAA, with a pH value of 5.6-5.8;

[0008] (2) cutting the cotyledons from the callus along the base of the embryo and inoculating them on a cotyledon-induced adventitious bud medium for culture to obtain adventitious bud clusters; the cotyledon-induced adventitious bud medium is: MS medium containing 1-3 mg / L 6-BA, 0.1 mg / L IBA, and 100 mg / L inositol, with a pH of 5.6-5.8;

[0009] (3) transferring the adventitious bud cluster to a regeneration bud induction medium for cultivation to obtain regeneration buds; the regeneration bud induction medium is: MS medium containing 1-3 mg / L 6-BA, 0.1 mg / L IBA, and 100 mg / L inositol, with a pH value of 5.6-5.8.

[0010] Preferably, in step (1), the immature embryos are obtained by disinfecting peach fruits 40-50 days after full flowering, cutting the peel, and peeling out the immature embryos.

[0011] Preferably, the variety of the peach tree is one or more of Xiahui No. 8, Zhongpan No. 17, and Ruiguang No. 39.

[0012] Preferably, in step (1), the immature embryo callus induction medium is: MS medium containing 0.25 mg / L 6-BA, 0.05 mg / L TDZ, 0.2 mg / L 2,4-D, and 0.1 mg / L NAA, with a pH value of 5.6-5.8.

[0013] Preferably, in step (1), the culture conditions are: temperature 24-26° C., relative humidity 50-60%, and dark treatment for 4 weeks.

[0014] Preferably, in step (2), the cotyledon-induced adventitious bud culture medium is: MS culture medium containing 2 mg / L 6-BA, 0.1 mg / L 1 BA, and 100 mg / L inositol, with a pH value of 5.6-5.8.

[0015] Preferably, in step (2), the culture conditions are: temperature 24-26° C., relative humidity 50-60%, light 16 h, dark 8 h, light intensity 2000-3000 lux, and the culture time is 4 weeks.

[0016] Preferably, in step (3), the regeneration bud induction medium is: MS medium containing 2 mg / L 6-BA, 0.1 mg / L IBA, and 100 mg / L inositol, with a pH value of 5.6-5.8.

[0017] Preferably, in step (3), the culture conditions are: temperature 24-26°C, relative humidity 50-60%, light 16h, dark 8h, light intensity 2000-3000 lux, and the culture time is 4 weeks.

[0018] Beneficial effects of the present invention:

[0019] The present invention provides a method for establishing a bud regeneration system using peach embryos. The method involves inducing buds by dark-culture of peach embryos 40 to 50 days after full flowering for four weeks. The enlarged cotyledons are then excised along the base of the embryos and inoculated onto a bud induction medium. This successfully regenerates adventitious bud clusters, which are then cultured to produce regenerated buds. This method facilitates future research into peach gene function and the rapid improvement of fruit trees and fruit quality, possessing significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : Feicheng peach embryo;

[0021] Figure 2 : Callus tissue on the immature embryo callus induction medium of AI treatment group;

[0022] Figure 3 : Adventitious buds induced from cotyledons of treatment groups 1-3 on adventitious bud culture medium;

[0023] Figure 4 : The culture results of Xiahui No. 8 in immature embryo callus induction medium;

[0024] Figure 5 : The culture results of Xiahui 8 in the cotyledon-induced adventitious bud culture medium;

[0025] Figure 6 : The culture results of Xiahui No. 8 in the regeneration bud induction medium;

[0026] Figure 7 :The culture results of Ruiguang 39 in immature embryo callus induction medium;

[0027] Figure 8 :The results of the culture of Ruiguang 39 in the cotyledon-induced adventitious bud culture medium;

[0028] Figure 9 :The culture results of Ruiguang 39 in regeneration bud induction medium;

[0029] Figure 10 : The results of the culture of Zhongpan 17 in immature embryo callus induction medium;

[0030] Figure 11 : The results of the culture of Zhongpan 17 in the cotyledon-induced adventitious bud culture medium;

[0031] Figure 12 : The culture results of Zhongpan 17 in regeneration shoot induction medium. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0033] As mentioned in the background, the current regeneration and genetic transformation efficiencies of peach materials are extremely low, preventing widespread production application. Therefore, the present invention selected the "Red Li Kuan Ye" Feicheng Buddha peach from the Feicheng Orchard in Tai'an City, Shandong Province, as experimental material. The experiment was conducted at the National Key Laboratory of Wheat Breeding at Shandong Agricultural University.

[0034] 1. Induction of immature embryo callus

[0035] Cut branches with fruits from the orchard in the field, put them into clean plastic bags and bring them back to the laboratory. Select fruits of uniform size from the branches and place them in a pot. Use a brush and detergent to clean the surface of the fruit for disinfection. In a sterilized empty culture dish, use a scalpel to cut the peel, remove the kernel, and carefully peel out the embryo with tweezers. Figure 1 As shown; the material was spread on the immature embryo callus induction medium and cultured to obtain callus tissue; the culture conditions were: temperature (25±1)°C, relative humidity 50-60%, and dark treatment for 4 weeks;

[0036] The experiment was divided into 9 treatment groups. The components of the immature embryo callus induction medium of each treatment group are shown in Table 1. Figure 2 As shown, Figure 2 A, B, C, D, E, F, G, H, and I correspond to treatment group A, treatment group B, treatment group C, treatment group D, treatment group E, treatment group F, treatment group G, treatment group H, and treatment group I in Table 1, respectively. Callus induction rate (%) = number of explants producing callus / total number of inoculated explants × 100%.

[0037] Table 1 Components of the culture medium for immature embryo callus induction in each treatment group

[0038]

[0039] Figure 2The results showed that all nine culture media induced callus, but the state of the callus induced by each treatment combination varied. Callus induced in treatments A, B, and C showed clearly visible, compact, and pale yellow granules, with callus induction rates of 67.24%, 63.33%, and 61.29%, respectively. Callus induced in treatments D, E, and F exhibited loose, granular structures that subsequently became densely granular during light culture, with callus induction rates of 74.14%, 66.13%, and 73.08%, respectively. Callus induced in treatments G and H were loose, delicate, and whitish, with callus induction rates of 75.47% and 68.89%, respectively. Callus induced in treatments I appeared clumpy, hard, and dull in color, exhibiting poor state, with a callus induction rate of 50.00%.

[0040] As can be seen, when the 6-BA concentration was fixed at 0.5 mg / L and TDZ was not added, increasing the dosage of 2,4-D and NAA decreased the callus induction rate from immature embryos. The optimal combination for inducing immature embryonic callus was Treatment A, achieving a callus induction rate of 67.24%. After the addition of TDZ, the callus induction rate decreased with increasing overall cytokinin concentration. Because both 6-BA and TDZ have cytokinin activity, their combined use may inhibit callus induction. Table 1 also shows that treatments with TDZ alone achieved higher callus induction rates than treatments with both TDZ and 6-BA, with the highest rate being achieved in Treatment G, which reached 75.47%. Increasing the TDZ concentration significantly decreased the callus induction rate, with the callus induction rate in Treatment I, at 2.0 mg / L, reaching only 50.00%.

[0041] In summary, the immature embryo callus induction medium of treatment group D was selected for subsequent experiments.

[0042] 2. Induction of adventitious buds from cotyledons

[0043] Cotyledons from the callus tissue of Treatment D were excised along the base of the embryo and inoculated onto a cotyledon-induced adventitious bud induction medium to produce adventitious bud clusters. Culture conditions included a temperature of 24-26°C, a relative humidity of 50-60%, a photoperiod of 16 hours of light and 8 hours of darkness, and a light intensity of 2000-3000 lux for four weeks. The experiment was divided into three treatment groups. The cotyledon-induced adventitious bud induction medium for each treatment group is shown in Table 2.

[0044] Table 2 Compositions of the culture medium for inducing adventitious buds from cotyledons of each treatment

[0045]

[0046] The culture results are as follows Figure 3As shown in the figure, A, B, and C correspond to treatment groups 1, 2, and 3 respectively, and D is the adventitious roots and regenerated shoots observed after 4 weeks of light culture on treatment combination 2. It can be seen that treatment group 2 has the best growth effect.

[0047] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0048] The test materials used in the examples of the present invention are all conventional test materials in the field and can be purchased through commercial channels. The MS solid culture medium used in the following examples and comparative examples of the present invention was purchased from Qingdao Haibo Biotechnology Co., Ltd., and 6-BA, TDZ, 2,4-D, NAA, IBA, and inositol were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. The 'Xiahui No. 8' and 'Zhongpan No. 17' used were from the Experimental Station of the South Campus of Shandong Agricultural University; 'Ruiguang No. 39' was from the Experimental Base of Hongmiao Community, Taishan District, Tai'an City, Shandong Province. The experimental site is the National Key Laboratory of Wheat Breeding of Shandong Agricultural University.

[0049] Example 1

[0050] A method for establishing a bud regeneration system using peach immature embryos, comprising the following steps:

[0051] (1) Cut fruit-bearing branches of Xiahui No. 8 from the field orchard 40-50 days after full bloom, place them in clean plastic bags and bring them back to the laboratory. Select fruits of uniform size from the branches and place them in a basin. Scrub the fruit surface with a brush and detergent for disinfection. In a sterilized empty Petri dish, cut the fruit peel with a scalpel, remove the kernel, and carefully peel out the embryo with tweezers.

[0052] The immature embryos are used as explants and inoculated into an immature embryo callus induction medium for culture to obtain callus tissue;

[0053] The medium for callus induction of immature embryos is: MS medium containing 0.25 mg / L 6-BA + 0.05 mg / L TDZ + 0.2 mg / L 2,4-D + 0.1 mg / L NAA, with a pH of 5.6-5.8;

[0054] The culture conditions were: temperature 24-26°C, relative humidity 50-60%, and dark treatment for 4 weeks.

[0055] The culture results are as follows Figure 4 As shown in the figure, a, b, and c are three repeated treatments of the same batch of materials.

[0056] (2) longitudinally incising the cotyledon of the callus obtained in step (1) along the base of the embryo, inoculating the callus on a cotyledon-induced adventitious bud culture medium, and culturing the callus to obtain an adventitious bud cluster;

[0057] The medium for inducing adventitious buds from cotyledons is: MS medium containing 2 mg / L 6-BA, 0.1 mg / L IBA, and 100 mg / L inositol, with a pH of 5.6-5.8;

[0058] Culture conditions: temperature 24-26°C, relative humidity 50-60%, light 16 hours, dark 8 hours, light intensity 2000-3000 lux, culture time is 4 weeks.

[0059] The results are as follows Figure 5 As shown, adventitious bud clusters can be seen on the culture medium.

[0060] (3) transferring the adventitious bud cluster obtained in step (2) to a regeneration bud induction medium for culturing to obtain regenerated buds;

[0061] The regeneration bud induction medium is: MS medium containing 2 mg / L 6-BA, 0.1 mg / L IBA, and 100 mg / L inositol, with a pH value of 5.6-5.8.

[0062] Culture conditions: temperature 24-26°C, relative humidity 50-60%, light 16 hours, dark 8 hours, light intensity 2000-3000 lux, culture time 4 weeks.

[0063] The experimental results are as follows Figure 6 As shown, regenerated shoots can be seen on the culture medium.

[0064] Example 2

[0065] A method for establishing a bud regeneration system using peach immature embryos, comprising the following steps:

[0066] (1) Cut fruit-bearing branches of Ruiguang 39 from the orchard 40-50 days after full bloom, place them in clean plastic bags, and bring them back to the laboratory. Select fruits of uniform size from the branches and place them in a basin. Use a brush and detergent to scrub the surface of the fruit clean for disinfection. In a sterilized empty Petri dish, cut the peel with a scalpel, remove the kernel, and carefully peel out the embryo with tweezers.

[0067] The immature embryos are used as explants and inoculated into an immature embryo callus induction medium for culture to obtain callus tissue;

[0068] The medium for callus induction of immature embryos is: MS medium containing 0.25 mg / L 6-BA + 0.05 mg / L TDZ + 0.2 mg / L 2,4-D + 0.1 mg / L NAA, with a pH of 5.6-5.8;

[0069] The culture conditions were: temperature 24-26°C, relative humidity 50-60%, and dark treatment for 4 weeks.

[0070] The culture results are as follows Figure 7 As shown in the figure, a, b, and c are three repeated treatments of the same batch of materials.

[0071] (2) longitudinally incising the cotyledon of the callus obtained in step (1) along the base of the embryo, inoculating the callus on a cotyledon-induced adventitious bud culture medium, and culturing the callus to obtain an adventitious bud cluster;

[0072] The medium for inducing adventitious buds from cotyledons is: MS medium containing 2 mg / L 6-BA, 0.1 mg / L IBA, and 100 mg / L inositol, with a pH of 5.6-5.8;

[0073] Culture conditions: temperature 24-26°C, relative humidity 50-60%, light 16 hours, dark 8 hours, light intensity 2000-3000 lux, culture time 4 weeks.

[0074] The results are as follows Figure 8 As shown, adventitious bud clusters can be seen on the culture medium.

[0075] (3) transferring the adventitious bud cluster obtained in step (2) to a regeneration bud induction medium for culturing to obtain regenerated buds;

[0076] The regeneration bud induction medium is: MS medium containing 2 mg / L 6-BA, 0.1 mg / L IBA, and 100 mg / L inositol, with a pH value of 5.6-5.8.

[0077] Culture conditions: temperature 24-26°C, relative humidity 50-60%, light 16 hours, dark 8 hours, light intensity 2000-3000 lux, culture time 4 weeks.

[0078] The experimental results are as follows Figure 9 As shown, regenerated shoots can be seen on the culture medium.

[0079] Example 3

[0080] A method for establishing a bud regeneration system using peach immature embryos, comprising the following steps:

[0081] (1) Cut fruit-bearing branches of Pan No. 17 from the field orchard 40-50 days after full bloom, place them in clean plastic bags and bring them back to the laboratory. Select fruits of uniform size from the branches and place them in a basin. Scrub the fruit surface with a brush and detergent for disinfection. In a sterilized empty Petri dish, cut the fruit peel with a scalpel, remove the kernel, and carefully peel out the embryo with tweezers.

[0082] The immature embryos are used as explants and inoculated into an immature embryo callus induction medium for culture to obtain callus tissue;

[0083] The medium for callus induction of immature embryos is: MS medium containing 0.25 mg / L 6-BA + 0.05 mg / L TDZ + 0.2 mg / L 2,4-D + 0.1 mg / L NAA, with a pH of 5.6-5.8;

[0084] The culture conditions were: temperature 24-26°C, relative humidity 50-60%, and dark treatment for 4 weeks.

[0085] The culture results are as follows Figure 10 As shown in the figure, a, b, and c are three repeated treatments of the same batch of materials.

[0086] (2) longitudinally incising the cotyledon of the callus obtained in step (1) along the base of the embryo, inoculating the callus on a cotyledon-induced adventitious bud culture medium, and culturing the callus to obtain an adventitious bud cluster;

[0087] The medium for inducing adventitious buds from cotyledons is: MS medium containing 2 mg / L 6-BA, 0.1 mg / L IBA, and 100 mg / L inositol, with a pH of 5.6-5.8;

[0088] Culture conditions: temperature 24-26°C, relative humidity 50-60%, light 16 hours, dark 8 hours, light intensity 2000-3000 lux, culture time 4 weeks.

[0089] The results are as follows Figure 11 As shown, adventitious bud clusters can be seen on the culture medium.

[0090] (3) transferring the adventitious bud cluster obtained in step (2) to a regeneration bud induction medium for culturing to obtain regenerated buds;

[0091] The regeneration bud induction medium is: MS medium containing 2 mg / L 6-BA, 0.1 mg / L IBA, and 100 mg / L inositol, with a pH value of 5.6-5.8.

[0092] Culture conditions: temperature 24-26°C, relative humidity 50-60%, light 16 hours, dark 8 hours, light intensity 2000-3000 lux, culture time 4 weeks.

[0093] The experimental results are as follows Figure 12 As shown, regenerated buds can be seen on the culture medium. A, B, C, and D in the figure are four repeated treatments of the same batch of materials.

[0094] Comparative Example 1

[0095] In this comparative example, the medium for inducing callus from immature embryos is: MS medium containing 0.25 mg / L 6-BA, with a pH value of 5.6-5.8; the rest is the same as in Example 3.

[0096] Comparative Example 2

[0097] In this comparative example, the medium for inducing callus from immature embryos is: MS medium containing 0.05 mg / L TDZ, with a pH value of 5.6-5.8; the rest is the same as in Example 3.

[0098] Comparative Example 3

[0099] In this comparative example, the medium for inducing callus from immature embryos is: MS medium containing 0.2 mg / L 2,4-D, with a pH value of 5.6-5.8; the rest is the same as in Example 3.

[0100] Comparative Example 4

[0101] In this comparative example, the culture medium for inducing callus from immature embryos is: MS culture medium containing 0.1 mg / L NAA, with a pH value of 5.6-5.8; the rest is the same as in Example 3.

[0102] Comparative Example 5

[0103] In this comparative example, the medium for inducing callus from immature embryos is MS medium with a pH value of 5.6-5.8; the rest is the same as in Example 3.

[0104] The callus induction rates and regeneration frequencies of Examples 1-3 are shown in Table 3. Regeneration frequency (%) = (number of regenerated bud explants / number of inoculated explants) x 100%.

[0105] The callus induction rates of Comparative Examples 1-5 are shown in Table 4.

[0106] Table 3 Callus induction rate and regeneration frequency of Examples 1-3

[0107] deal with Callus induction rate (%) Regeneration frequency (%) Example 1 58.2 4.3 Example 2 62.5 9.2 Example 3 80.9 14.5

[0108] Table 4 Callus induction rate of Example 3 and Comparative Examples 1-5

[0109] deal with Callus induction rate (%) Example 3 80.9 Comparative Example 1 30.57 Comparative Example 2 34.38 Comparative Example 3 26.94 Comparative Example 4 23.25 Comparative Example 5 12.14

[0110] As can be seen from Table 3, the callus induction rate of Zhongpan No. 17 can reach 80.9%, and the regeneration frequency can reach 14.5%. As can be seen from Table 4, the combination of 6-BA, TDZ, 2,4-D, and NAA in the immature embryo callus induction medium of the present invention has a synergistic effect on improving the callus induction rate.

[0111] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A method for establishing a bud regeneration system using peach immature embryos, characterized in that: The following steps are involved: (1) taking peach immature embryos as explants, inoculating them into an immature embryo callus induction medium for culture, and obtaining callus tissue; the immature embryo callus induction medium is: MS medium containing 0.25 mg / L 6-BA, 0.05 mg / L TDZ, 0.2 mg / L 2,4-D, and 0.1 mg / L NAA, with a pH value of 5.6-5.8; (2) cutting the cotyledons from the callus along the base of the embryo and inoculating them on a cotyledon-induced adventitious bud medium to obtain an adventitious bud cluster; the cotyledon-induced adventitious bud medium is: MS medium containing 2 mg / L 6-BA, 0.1 mg / L IBA, and 100 mg / L inositol, with a pH of 5.6-5.8; (3) transferring the adventitious bud cluster to a regeneration bud induction medium for culturing to obtain regenerated buds; the regeneration bud induction medium is: MS medium containing 2 mg / L 6-BA, 0.1 mg / L IBA, and 100 mg / L inositol, with a pH value of 5.6-5.8; The variety of the peach tree is Zhongpan No.

17.

2. The method for establishing a bud regeneration system utilizing peach immature embryos according to claim 1, wherein In step (1), the young embryos are obtained by disinfecting peach fruits 40-50 days after full flowering, cutting the peel and peeling out the young embryos.

3. The method for establishing a bud regeneration system utilizing peach immature embryos according to claim 1, wherein In step (1), the culture conditions are: temperature 24-26° C., relative humidity 50-60%, and dark treatment for 4 weeks.

4. The method for establishing a bud regeneration system utilizing peach immature embryos according to claim 1, wherein In step (2), the culture conditions are: temperature 24-26° C., relative humidity 50-60%, light 16 h, dark 8 h, light intensity 2000-3000 lux, and the culture time is 4 weeks.

5. The method for establishing a bud regeneration system utilizing peach immature embryos according to claim 1, wherein In step (3), the culture conditions are: temperature 24-26° C., relative humidity 50-60%, light 16 h, dark 8 h, light intensity 2000-3000 lux, and the culture time is 4 weeks.

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