Method for establishing a high-efficiency regeneration system of autumn pear leaf

Abstract

This invention discloses a method for establishing a high-efficiency regeneration system for *Pyrus pyrifolia* leaves, belonging to the field of plant tissue culture technology. The method includes: spreading a layer of sorbitol solution on the surface of a culture medium; inoculating *Pyrus pyrifolia* seedlings into the medium and placing them in a dark environment for etiolation treatment; taking etiolated leaves from the *Pyrus pyrifolia* seedlings and inoculating them in a regeneration medium for dark culture, then transferring them to light for further culture to produce adventitious buds; after the adventitious buds reach the target length, cutting off their lateral buds and placing them on a subculture medium to obtain regenerated seedlings. This invention, by adding sorbitol solution before dark treatment, can significantly shorten the time to obtain etiolated material to 7 days, and the etiolated plants are in better condition after three weeks; furthermore, by placing *Pyrus pyrifolia* tissue culture seedlings in a dark environment for dark culture, the physiological state of the tissue culture seedlings is altered, and the obtained etiolated leaves are inoculated into the regeneration medium, improving the regeneration efficiency, and the material acquisition is not affected by the season.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a method for establishing a high-efficiency regeneration system for leaves of *Pyrus pyrifolia*. Background Technology

[0002] Pears are deciduous fruit trees belonging to the genus *Pyrus* L. of the family Rosaceae. They are cultivated and distributed in more than 80 countries worldwide and have a long history of cultivation. China boasts abundant pear varietal resources, with 22 widely recognized species, 13 of which are native to China, making China the country with the richest pear varietal resources in the world. The main cultivated varieties in China include white pear, sand pear, autumn pear, and Xinjiang pear.

[0003] The Qiuzi pear (Pyrus ussuriensis Maxim.), also known as the sand pear, sour pear, or mountain pear, originated in Northeast my country and is distributed in Northeast, North, and Northwest China. Its main propagation method is grafting, but the availability of high-quality scions is limited, which affects the growth and development of the variety and the quality of the fruit.

[0004] Plant tissue culture ensures uniform rootstock seedlings, facilitating rapid propagation. Pear tissue culture has been practiced for over 20 years, and significant progress has been made in establishing regeneration systems. However, problems such as difficulty in rooting and low regeneration efficiency still exist, severely hindering subsequent genetic transformation research. Therefore, establishing a highly efficient leaf regeneration system for *Pyrus pyrifolia* has become crucial. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for establishing a high-efficiency regeneration system for pear leaves.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a method for establishing a high-efficiency regeneration system for autumn pear leaves, comprising: The Qiuzi pear was placed in a dark environment to undergo chlorosis treatment; the chlorotic leaves of the Qiuzi pear were inoculated into a regeneration medium and cultured in the dark, and then transferred to light for culture to produce adventitious buds; after the adventitious buds grew to the target length, the lateral buds were cut off and cultured on a subculture medium to obtain regenerated seedlings.

[0007] Furthermore, a layer of sorbitol solution was spread on the surface of the culture medium, and the pear seedlings were inoculated into the culture medium and placed in the dark for etiolation treatment. The etiolated leaves of the pear were taken and inoculated into the regeneration culture medium for dark culture, and then transferred to light for culture to produce adventitious buds. After the adventitious buds grew to the target length, the lateral buds were cut off and placed on the subculture medium for culture to obtain regenerated seedlings.

[0008] Furthermore, a layer of sorbitol solution is spread on the surface of the culture medium, wherein the culture medium is MS + 0.1 mg / L IBA + 0.5 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar, and the pH is 5.75-5.8.

[0009] The concentration of the sorbitol solution is any value between 5 and 10 mg / L.

[0010] Furthermore, the regeneration medium was NN69 + 3.0 mg / L TDZ + 0.3 mg / L IBA + 30 g / L sucrose + 7 g / L agar, with a pH of 5.8.

[0011] Furthermore, the subculture medium was MS + 0.1 mg / L IBA + 0.5 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar, with a pH of 5.75-5.8.

[0012] Furthermore, the target length is any value between 0.5 and 1.2 cm.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: the addition of sorbitol solution before dark treatment can significantly shorten the time to obtain etiolated materials to 7 days, and the etiolated plants are in better condition after three weeks, effectively solving the problems of long cycle and unstable material condition in traditional dark treatment.

[0014] By placing the tissue culture seedlings of Pyrus pyrifolia in a dark environment for dark culture, the physiological state of the tissue culture seedlings was changed. The obtained yellowed leaves were then inoculated into the regeneration medium, which improved the regeneration efficiency. Moreover, the material was not affected by the season, and the process was efficient, fast, simple, and not subject to requirements on leaf variety or leaf age. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The yellowing of leaves in tissue culture seedlings of *Pyrus pyrifolia* is shown in the following figures: A represents aseptic tissue culture seedlings of *Pyrus pyrifolia*; B represents aseptic tissue culture seedlings of *Pyrus pyrifolia* after two to four weeks of dark treatment; C represents aseptic tissue culture seedlings of *Pyrus pyrifolia* after 7 days of sorbitol treatment; D represents aseptic tissue culture seedlings of *Pyrus pyrifolia* after 3 weeks of sorbitol treatment; and E represents usable yellowed plants.

[0017] Figure 2 Regeneration rate of conventional and yellowed pear leaves.

[0018] Figure 3 The average number of regenerated buds per leaf of conventional and yellowed pear leaves. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0020] The present invention discloses a method for establishing a high-efficiency regeneration system for autumn pear leaves, comprising the following steps: (1) Preparation of etiolated explant material of Pyrus pyrifolia: Place sterile tissue culture seedlings of Pyrus pyrifolia in a dark environment, and etiolated leaves are obtained in two to four weeks.

[0021] Preferably, before placing the sterile tissue culture seedlings into a dark environment, a layer of 5 mg / L sorbitol solution is spread evenly on the surface of the culture medium. Dark treatment for 7 days yields etiolated, usable plants, and the condition of the etiolated, usable plants after three weeks of treatment is better than that of plants treated in the dark alone. In another embodiment, a 10 mg / L sorbitol solution can also be used, or any concentration between 5 and 10 mg / L can be selected; these will not be elaborated further here.

[0022] (2) Differentiation and regeneration culture: The yellowed leaves of *Pyrus pyrifolia* were inoculated in regeneration medium and cultured in the dark for 15 days, and then transferred to light for culture to produce adventitious buds. The regeneration medium was based on NN69 medium, with the addition of 3.0 mg / L LTDZ, 0.3 mg / L IBA, 30 g / L sucrose, and 7 g / L agar.

[0023] (3) Propagation by subculture of adventitious buds: When the adventitious buds grow to 0.5-1.2cm, cut off their lateral buds and place them on the subculture medium to obtain regenerated seedlings.

[0024] In step (1), the amount of sorbitol solution used for spreading is 1 mL, which is sufficient to completely wet the surface of the culture medium.

[0025] In step (2), the regeneration medium is NN69 + 3.0 mg / L TDZ + 0.3 mg / L IBA + 30 g / L sucrose + 7 g / L agar, with a pH of 5.8.

[0026] In step (3), the subculture medium is MS + 0.1 mg / L IBA + 0.5 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar, with a pH of 5.75-5.8.

[0027] Example 1: Preparation of etiolated explant material from *Pyrus pyrifolia* The preparation of etiolated explants of *Pyrus pyrifolia* using the method provided in this invention is as follows: Sterile tissue culture seedlings of Pyrus pyrifolia were placed in a dark environment with a culture room temperature of 24±2℃, and yellowing leaves were obtained in two to four weeks.

[0028] Example 2: Regeneration of conventional and yellowed pear leaves Leaves of both conventional and etiolated *Pyrus pyrifolia* were inoculated into regeneration medium consisting of NN69 as the basal medium, supplemented with 3.0 mg / L TDZ + 0.3 mg / L IBA, 30 g sucrose and 7 g agar per liter of medium, and the pH was adjusted to 5.8. After 15 days of dark incubation, the medium was transferred to light incubation at a temperature of 25 ± 2 ℃ and a photoperiod of 16 h / d for 12 days.

[0029] Example 3: Propagation through subculture of adventitious buds Once the adventitious buds have grown to approximately 0.5-1.2 cm, their lateral buds are cut off and placed on a subculture medium. The subculture medium is a conventional subculture medium used for the proliferation of *Pyrus pyrifolia*, with the following formula: MS medium + 0.1 mg / L IBA + 0.5 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar, pH 5.75-5.8, autoclaved for 15 min before use.

[0030] Example 4: Sorbitol pretreatment accelerates the acquisition of xanthated materials Before placing aseptic tissue culture seedlings of *Pyrus pyrifolia* in darkness, a layer of 1 mL of 5 mg / L sorbitol solution was evenly spread on the surface of the culture medium. The seedlings were then incubated in darkness at a temperature of 25 ± 2 °C. (See also...) Figure 1 The results showed that usable etiolated plants could be obtained after 7 days of treatment, which was shorter than the traditional dark treatment (2-4 weeks). After three weeks of cultivation, the etiolated plants in the sorbitol pretreatment group had fully expanded leaves, uniform etiolation, and vigorous growth, and were in significantly better condition than the dark treatment group alone.

[0031] Comparing the leaf regeneration in Example 3, a one-way ANOVA was performed on the statistical data of leaf regeneration rate and average number of regenerated shoots per leaf. The homogeneity of variance test showed P>0.05, satisfying the homogeneity of variance requirement. A one-way ANOVA test was then performed on the results, with P<0.05. Subsequently, the LSD method was used for multiple comparisons, and the results are as follows: Figure 2 and Figure 3 As shown (regeneration rate = number of regenerated leaves / number of inoculated leaves × 100%; average number of regenerated buds per leaf = total number of regenerated buds / number of regenerated leaves).

[0032] A leaf regeneration system for *Pyrus pyrifolia* was established using the above-mentioned techniques. The system is simple to operate, achieves a 100% regeneration rate, and has a regeneration coefficient of 9-14, demonstrating high regeneration efficiency. This system can provide a reference for exploring regeneration systems for other pear varieties, thereby solving the problem of generally low pear regeneration rates. Simultaneously, pretreatment with sorbitol solution accelerates the acquisition of etiolated materials; this method does not affect subsequent leaf regeneration efficiency.

[0033] The above embodiments are only used to illustrate the design concept and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. The protection scope of the present invention is not limited to the above embodiments. Therefore, all equivalent changes or modifications made based on the principles and design ideas disclosed in the present invention are within the protection scope of the present invention.

Claims

1. A method for establishing a high-efficiency regeneration system for autumn pear leaves, characterized in that, include: The Qiuzi pear was placed in a dark environment to undergo chlorosis treatment; the chlorotic leaves of the Qiuzi pear were inoculated into a regeneration medium and cultured in the dark, and then transferred to light for culture to produce adventitious buds; after the adventitious buds grew to the target length, the lateral buds were cut off and cultured on a subculture medium to obtain regenerated seedlings.

2. The method according to claim 1, characterized in that, The process of placing the autumn pear in a dark environment for yellowing also includes: A layer of sorbitol solution was spread on the surface of the culture medium, and the pear seedlings were inoculated into the culture medium and placed in a dark environment for etiolation treatment.

3. The method according to claim 2, characterized in that, The step involves spreading a layer of sorbitol solution on the surface of the culture medium, wherein the culture medium is MS + 0.1 mg / L IBA + 0.5 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar, with a pH of 5.75-5.8; The concentration of the sorbitol solution is any value between 5 and 10 mg / L.

4. The method according to claim 1, characterized in that, The regeneration medium was NN69 + 3.0 mg / L TDZ + 0.3 mg / L IBA + 30 g / L sucrose + 7 g / L agar, with a pH of 5.

8.

5. The method according to claim 1, characterized in that, The subculture medium was MS medium containing 0.1 mg / L IBA, 0.5 mg / L 6-BA, 30 g / L sucrose, and 7 g / L agar, with a pH of 5.75-5.

8.

6. The method according to claim 1, characterized in that, The target length is any value between 0.5 and 1.2 cm.

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