A method for improving pear leaf regeneration efficiency and rapid propagation

Abstract

The application discloses a method for improving leaf regeneration efficiency and rapid propagation of pears, and comprises the following steps: (1) taking full and mature seeds of the pears to perform disinfection treatment; (2) after peeling the seeds, the seeds are inoculated in an agar solid culture medium to perform culture; (3) after the leaf of the pear is fully unfolded, the top and the base of the leaf are cut off under a sterile condition, and the leaf is inoculated in a regeneration culture medium to perform dark culture; (4) after the dark culture is performed for 30±1 days, the leaf is cultured under a light and dark culture condition to obtain adventitious buds; and (5) the regenerated adventitious buds are cut off and inoculated in a subculture medium to perform rapid propagation and expansion. The application obtains regenerated adventitious buds by using the leaf of the pear, improves the leaf regeneration efficiency of the pear, selects a suitable dark culture time, solves the problem of low leaf regeneration efficiency of the pear, and provides a stable and efficient leaf regeneration system for subsequent related research on induction of the adventitious buds by using the leaf as an explant.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a method for adventitious bud regeneration and rapid propagation using pear leaves as explants. Background Technology

[0002] Pears, belonging to the genus Pyrus in the family Rosaceae, are an important fruit in my country. my country boasts abundant pear germplasm resources, with the world's major cultivated varieties—Qiuzi pear, Bai pear, and Sha pear—all originating in my country. There are as many as 130 cultivable varieties. Pear trees are highly adaptable to various soil types, have a long lifespan, and have a long history of economic use, resulting in widespread cultivation. However, pear trees have a long juvenile stage, and propagation is primarily done through seedling cultivation and grafting, which is time-consuming, has a low survival rate, and makes it difficult to obtain superior hybrids through traditional breeding methods.

[0003] The development of plant biotechnology, especially tissue culture and genetic engineering, has enabled the rapid proliferation of superior varieties through asexual reproduction via plant tissue culture, which is not limited by season and can be achieved in a short period of time. This provides a new pathway for the targeted improvement of pear production traits and the cultivation of varieties with excellent overall quality. Studies have shown that the regeneration efficiency of pear cotyledons as explants is higher than that of other parts. Although tissue culture technology using cotyledons as explants has been applied to pears, the regeneration efficiency has remained low. Establishing an efficient regeneration system is of great significance for subsequent genetic transformation work in pears. Summary of the Invention

[0004] This invention provides a method to improve the regeneration efficiency and rapid proliferation of pear leaves, and establishes a stable and efficient regeneration system and a subgeneration propagation system, laying a certain foundation for genetic transformation.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] A method for improving the regeneration efficiency and rapid proliferation of pear leaves, the method comprising the following steps:

[0007] (1) Take plump and ripe pear seeds and disinfect them;

[0008] (2) After the sterilized seeds are peeled, they are inoculated into agar solid medium for culture;

[0009] (3) After the pear leaves have fully unfolded, under sterile conditions, cut off the top and base of the cotyledons, inoculate them into the regeneration medium, and carry out dark culture.

[0010] The regeneration medium is formulated as follows: NN69 medium + 6-BA 1.0 mg / L + NAA 0.5 mg / L + TDZ 1.0 mg / L + sorbitol 40 g / L + plant gel 2.5 g / L, pH 5.80-5.85;

[0011] (4) After dark culture for 30±1 days, it is cultured under light and dark conditions to obtain adventitious buds;

[0012] (5) Cut off the regenerated adventitious buds and inoculate them into the subculture medium for rapid proliferation and propagation.

[0013] As a preferred technical solution: the disinfection process in step (1) is as follows: the seeds are first washed with 75% alcohol for 1 min in a clean bench, then rinsed with sterile water 3-4 times, then washed with 2.5% sodium hypochlorite for 30 min, rinsed with sterile water 8-10 times, and after being cleaned with disinfectant, soaked in sterile water overnight, and inoculated on the second day.

[0014] As a preferred technical solution: In step (2), after the disinfected seeds are peeled off in a clean bench, they are inoculated in 7.5 g / L agar solid medium and cultured. They are first cultured in the dark for 3 days, and then transferred to a light intensity of 2000 lx and a temperature of 25 ± 2℃ for 16 h / 8 h light and dark culture.

[0015] As a preferred technical solution: the light and dark culture conditions in step (4) are: light intensity 2000 lx, temperature 25±2℃ for 16h / 8h light and dark culture.

[0016] As a preferred technical solution, the dark culture time in step (4) is preferably 30 days.

[0017] As a preferred technical solution: the formula of the subculture medium in step (5) is: MS medium + 6-BA 2.0 mg / L + IBA 0.2 mg / L + sucrose 30 g / L + agar 7.5 g / L, pH 5.80-5.85.

[0018] As a preferred technical solution: the pear is the 'Fengshui' pear.

[0019] The beneficial effects of this invention are as follows:

[0020] This invention determined the optimal dark culture time and explored the regeneration effects of explants from different varieties and sources using different combinations of growth regulators. A stable and efficient regeneration system was obtained through comparison, achieving a regeneration rate of 88.33% and a regenerated adventitious bud number of 4.28. Subculture propagation showed rapid growth, with an average proliferation coefficient of 3.33 and a plant height of 3.80 cm. Attached Figure Description

[0021] Figure 1 The regeneration effect of propagating different pear varieties.

[0022] Figure 2 The growth of 'Fengshui' pear leaves on a culture medium with the optimal hormone ratio.

[0023] Figure 3 The growth of adventitious buds regenerated from the leaves of 'Fengshui' pear after inoculation onto the optimal subculture medium. Detailed Implementation

[0024] This invention focuses on the experimental design regarding the ratio of growth regulators in the culture medium and the dark incubation time. To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0025] Example 1: A method for improving the regeneration efficiency and rapid propagation of leaves in 'Fengshui' pear trees

[0026] The screening process of this method:

[0027] 1. Plant materials

[0028] The material used was laboratory-grown 'Fengshui' pear seeds.

[0029] 2. Test Methods

[0030] (1) Take plump and mature pear seeds, disinfect them, and wash them with 75% alcohol for 1 min in a clean bench, then rinse them with sterile water 4 times, then wash them with 2.5% sodium hypochlorite for 30 min, rinse them with sterile water 10 times, and after cleaning them with disinfectant, soak them in sterile water overnight and inoculate them on the second day.

[0031] (2) After the seeds were sterile and peeled, they were inoculated with 7.5 g / L agar solid medium and cultured. They were first cultured in the dark for 3 days, and then transferred to light intensity of 2000 lx and temperature of (25±2)℃ for 16h / 8h light and dark culture.

[0032] (3) After the pear leaves have fully expanded, the top and base of the cotyledons are cut off under aseptic conditions and inoculated into regeneration media with different hormone ratios. NN69 medium is used as the basal medium, 40 g / L sorbitol is used as the sugar source, 2.5 g / L plant gel is used as the solidifying agent, and the pH is 5.80-5.85. The plant hormones used are cytokinin 6-BA, TDZ and auxin NAA. The screening concentrations of 6-BA hormone are set to 1.0 mg / L, 3.0 mg / L and 5.0 mg / L, the screening concentrations of NAA are set to 0.1 mg / L, 0.5 mg / L and 1.0 mg / L, and the screening concentrations of TDZ are set to 0.5 mg / L, 1.0 mg / L and 1.5 mg / L, for a total of 27 formulations. The formulations are then cultured in the dark for 30 days. The results in Table 1 show that the hormone ratio of 5,6-BA 1.0 mg / L + NAA 0.5 mg / L + TDZ 1.0 mg / L is suitable for the regeneration of 'Fengshui' cotyledons, with a regeneration rate of 88.33% and a regeneration adventitious bud number of 4.28.

[0033] Table 1 Culture media with different hormone ratios

[0034] Treatment 6-BA (mg / mL) NAA (mg / mL) TDZ (mg / mL) 1 1.0 0.1 0.5 2 1.0 0.1 1.0 3 1.0 0.1 1.5 4 1.0 0.5 0.5 5 1.0 0.5 1.0 6 1.0 0.5 1.5 7 1.0 1.0 0.5 8 1.0 1.0 1.0 9 1.0 1.0 1.5 10 3.0 0.1 0.5 11 3.0 0.1 1.0 12 3.0 0.1 1.5 13 3.0 0.5 0.5 14 3.0 0.5 1.0 15 3.0 0.5 1.5 16 3.0 1.0 0.5 17 3.0 1.0 1.0 18 3.0 1.0 1.5 19 5.0 0.1 0.5 20 5.0 0.1 1.0 21 5.0 0.1 1.5 22 5.0 0.5 0.5 23 5.0 0.5 1.0 24 5.0 0.5 1.5 25 5.0 1.0 0.5 26 5.0 1.0 1.0 27 5.0 1.0 1.5

[0035] Note: Different letter labels indicate significant differences (p<0.05).

[0036] Table 2. Effects of different hormone ratios on adventitious bud regeneration of cotyledons in 'Fengshui' cultivar.

[0037] Treatment Cotyledon regeneration rate (%) Number of adventitious buds of cotyledons 1 33.33±9.61ghi 1.36±0.64ijk 2 40.67±3.79efg 1.44±1.11ij 3 43.00±10.82def 1.88±0.12efg 4 63.67±6.11b 2.53±0.26b 5 88.33±3.78a 4.28±0.26a 6 58.33±6.66bc 2.32±0.24bc 7 47.00±8.18de 2.36±0.28bc 8 48.33±7.02de 2.23±0.17cd 9 45.67±2.08de 2.06±0.06de 10 20.00±5.00klmn 1.99±0.07ef 11 29.67±6.11hijk 2.21±0.11cd 12 23.67±6.81ijklm 1.71±0.15gh 13 35.67±4.04fgh 1.86±0.09efg 14 50.33±4.04cd 1.95±0.05efg 15 27.67±2.52hijk 1.72±0.11gh 16 25.67±3.79ijkl 1.72±0.09gh 17 30.33±5.03hij 1.81±0.15fg 18 22.67±2.08jklm 1.53±0.07hi 19 20.00±4.00klmn 1.27±0.07jkl 20 22.00±2.65jklm 1.11±0.09lm 21 21.00±4.36jklm 1.20±0.07klm 22 23.67±2.52ijklm 1.38±0.05ijk 23 28.67±3.22hijk 1.48±0.05ij 24 17.67±2.52lmn 1.00m 25 15.00±1.00mn 1.18±0.06klm 26 16.33±2.08lmn 1.00m 27 11.00±1.00n 1.00m

[0038] Note: Different letter labels indicate significant differences (p<0.05).

[0039] (4) After 30 days of dark culture, the culture was transferred to 16h / 8h light and dark culture with a light intensity of 2000 lx and a temperature of (25±2)℃ to obtain adventitious buds.

[0040] (5) After selecting the optimal culture formula, select the appropriate dark culture time. Inoculate the leaves of 'Fengshui' pear into the optimal culture system (NN69 medium + 6-BA 1.0 mg / L + NAA 0.5 mg / L + TDZ 1.0 mg / L + sorbitol 40 g / L + plant gel 2.5 g / L, pH 5.80-5.85). After dark culture for 0 d, 7 d, 14 d, 21 d, 30 d and 45 d respectively, transfer to light and dark culture for 16 h / 8 h, light intensity of 2000 lx, temperature (25±2) ℃ for culture to improve the regeneration system.

[0041] Table 3. Effects of dark incubation time on cotyledon regeneration in 'Fengshui' cultivar.

[0042] Dark cultivation time Regeneration rate (%) Number of regenerated adventitious buds Dark incubation time Regeneration rate (%) Number of regenerated buds 0 0f 0f 7 30.26±4.56e 1.38±0.16e 14 39.71±1.22d 1.91±0.13d 21 73.48±2.85b 3.33±0.21b 30 91.43±1.52a 4.23±0.23a 45 62.25±2.58c 2.43±0.21c

[0043] Note: Different letter labels indicate significant differences (p<0.05).

[0044] (6) At the same time, adventitious buds regenerated from different pear varieties ('Fengshui' pear, 'Cuiguan' pear, and Qiuzi pear) were inoculated into the same subculture medium (MS medium + 6-BA 2.0 mg / L + IBA 0.2 mg / L + sucrose 30 g / L + agar 7.5 g / L, pH 5.80-5.85) for culture. Figure 1 It can be seen that the subculture system (MS medium + 6-BA 2.0 mg / L + IBA 0.2 mg / L + sucrose 30 g / L + agar 7.5 g / L) is suitable for 'Fengshui' pear, with a relatively fast growth rate, an average proliferation coefficient of 3.33, and a plant height of 3.80.

[0045] The results from the implementation examples show that the regeneration medium formula: NN69 medium + 6-BA 1.0 mg / L + NAA 0.5 mg / L + TDZ 1.0 mg / L + sorbitol 40 g / L + plant gel 2.5 g / L is a suitable hormone ratio for the regeneration of 'abundant water' pear leaves, with a regeneration rate of up to 88.33% and a regenerated adventitious bud number of up to 4.28 (e.g., ...). Figure 2 Subculture medium system: MS medium + 6-BA 2.0 mg / L + IBA 0.2 mg / L + sucrose 30 g / L + agar 7.5 g / L is suitable for the subculture of regenerated shoots of 'Fengshui' pear. It exhibits rapid growth, with an average proliferation coefficient of 3.33 and a plant height of 3.80 cm (e.g., ...). Figure 3 This demonstrates that the present invention establishes a stable and efficient regeneration system, laying the foundation for genetic transformation.

Claims

1. A method for improving the regeneration efficiency and rapid proliferation of pear leaves, characterized in that: The method includes the following steps: (1) Take plump and ripe pear seeds and disinfect them; (2) After the sterilized seeds are peeled, they are inoculated into agar solid medium for culture; (3) After the pear leaves have fully unfolded, under sterile conditions, cut off the top and base of the cotyledons, inoculate them into the regeneration medium, and carry out dark culture. The regeneration medium is formulated as follows: NN69 medium + 6-BA 1.0 mg / L + NAA 0.5 mg / L + TDZ 1.0 mg / L + sorbitol 40 g / L + plant gel 2.5 g / L, pH 5.80-5.85; (4) After dark culture for 30±1 days, it is cultured under light and dark conditions to obtain adventitious buds; (5) Cut off the regenerated adventitious buds and inoculate them into the subculture medium for rapid proliferation and propagation.

2. The method according to claim 1, characterized in that: The disinfection process described in step (1) is as follows: the seeds are first washed with 75% alcohol for 1 minute in a clean bench, then rinsed with sterile water 3-4 times, then washed with 2.5% sodium hypochlorite for 30 minutes, rinsed with sterile water 8-10 times, and after being cleaned with disinfectant, soaked in sterile water overnight, and inoculated on the second day.

3. The method according to claim 1, characterized in that: In step (2), after the disinfected seeds are peeled off in a clean bench, they are inoculated into 7.5 g / L agar solid medium and cultured in the dark for 3 days, and then transferred to a light intensity of 2000 lx and a temperature of 25 ± 2℃ for 16 h / 8 h light and dark culture.

4. The method according to claim 1, characterized in that: The light and dark culture conditions described in step (4) are: light intensity of 2000 lx and temperature of 25 ± 2℃ for 16 h / 8 h light and dark culture.

5. The method according to claim 1, characterized in that: The dark culture time mentioned in step (4) is 30 days.

6. The method according to claim 1, characterized in that: The subculture medium in step (5) is formulated as follows: MS medium + 6-BA 2.0 mg / L + IBA 0.2 mg / L + sucrose 30 g / L + agar 7.5 g / L, pH 5.80-5.

85.

7. The method according to claim 1, characterized in that: The pear mentioned is the 'Fengshui' pear.

Images