A tissue culture method for inducing adventitious bud regeneration by using peach leaf as explant

Abstract

The application discloses a tissue culture method for inducing adventitious bud regeneration by taking peach leaf as an explant. The application solves the problem of difficult peach adventitious bud regeneration, and the adventitious bud induction medium of the application successfully induces adventitious buds from the cotyledon of mature peach seeds. After bud elongation culture and adventitious root induction, the complete regenerated plant tissue culture seedlings are formed, and further through seedling hardening, normal growth seedlings are obtained. The application takes the cotyledon of mature peach seeds as the explant, has the advantages of simple disinfection and sterilization method, low cost, easy explant sampling, no season limitation, annual repetition, high regeneration efficiency, and provides technical support for the construction of a stable genetic transformation system of peach, is the basis of peach molecular breeding, and has wide application prospect in variety improvement.

Description

Technical Field

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

[0002] The peach (Prunus persica), native to China, is a perennial woody plant belonging to the subgenus Prunus of the genus Prunus in the family Rosaceae. It is highly valued by consumers for its high ornamental value, excellent fruit flavor, and rich nutritional content. Molecular breeding technology can significantly accelerate the selection of high-quality peach varieties, thereby meeting the growing market demand for quality traits. However, as a perennial woody fruit tree, the genetic transformation of the peach has always been a recognized technical challenge. Currently, we have successfully established stable genetic transformation systems for peach roots and callus tissue, but the difficulty of adventitious bud regeneration remains a bottleneck restricting the establishment of genetic transformation systems for complete peach plants. Therefore, establishing an efficient adventitious bud regeneration system will provide important technical support for the construction of stable genetic transformation systems for peaches, thereby promoting the widespread application of molecular-assisted breeding technology in the cultivation of high-quality peach varieties. Summary of the Invention

[0003] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of this invention is to provide a tissue culture method for inducing adventitious bud regeneration using peach leaves as explants. The culture medium and method of this invention can realize the regeneration of adventitious buds from peach leaf explants, and the materials are easy to obtain, not limited by time, and have high regeneration efficiency.

[0004] The present invention provides a tissue culture method for inducing adventitious bud regeneration using peach leaves as explants, comprising the following steps:

[0005] a. Take mature and intact peach seeds, disinfect them, dry the surface moisture, cut off both ends of the seeds to remove the embryo, peel off the seed coat, and obtain cotyledons with wounds at both ends as explants;

[0006] b. Place the cotyledon explants on an adventitious bud induction medium to induce adventitious bud formation; the adventitious bud induction medium is: MS + 4 mg / L 6-BA + 0.1 mg / L IBA + 30 g / L sucrose + 8 g / L agar, pH = 5.8;

[0007] c. Once the adventitious buds have grown to 0.5-1 cm in length, cut them off from the cotyledon explants and transfer them to an adventitious bud elongation medium for culture, allowing the adventitious buds to elongate and grow. The adventitious bud elongation medium is: MS + 0.5 mg / L 6-BA + 0.1 mg / L IBA + 30 g / L sucrose + 8 g / L agar, pH = 5.8.

[0008] d. After the adventitious buds grow to 3-5 cm in length, cut them off from the base of the bud segment and transfer them to an adventitious root induction medium to induce the occurrence and growth of adventitious roots. The adventitious root induction medium is: MS + 2 mg / L 6-BA + 30 g / L sucrose + 8 g / L agar, pH = 5.8. After the seedlings have grown into complete tissue culture seedlings, harden them off in a timely manner.

[0009] e. Transplant the hardened tissue culture seedlings into nutrient soil for further cultivation.

[0010] Preferably, the peach is of the 'Zaoyoutao' variety.

[0011] Preferably, the disinfection process involves soaking peach seeds in a 3% hypochlorous acid aqueous solution for 30 minutes, rinsing the seeds with sterile water 6-8 times, and then soaking them for 12 hours.

[0012] Preferably, the hardening-off process involves, after the tissue culture seedlings have grown into complete seedlings with well-developed root systems, injecting 3 mL of sterile water into the culture bottle to cover the entire surface of the culture medium, opening the bottle cap, and hardening off the seedlings in the tissue culture room. After 5 days of hardening-off, the tissue culture seedlings are removed, and the culture medium on the roots is washed off.

[0013] Preferably, the nutrient soil is prepared by mixing perlite and humus in a volume ratio of 1:2.

[0014] Preferably, the cultivation conditions in step bd are: light intensity 800-1200 Lux, light cycle 16h light / 8h dark, temperature 24℃-26℃, and relative humidity 65%.

[0015] Preferably, the cultivation conditions in step e are: light intensity 1200-1600 Lux, light cycle 16h light / 8h dark, temperature 22℃-24℃, and relative humidity 65%.

[0016] The present invention has the following beneficial effects:

[0017] In the process of adventitious bud induction using cotyledons of mature peach seeds as explants, the formulation of the adventitious bud induction culture medium and the selection of explants are crucial. Using this medium, adventitious buds were successfully induced from the cotyledons of mature peach seeds, solving the problem of difficult adventitious bud regeneration in peaches. After adventitious bud elongation culture and rooting, de novo regenerated tissue culture seedlings can be obtained, providing technical support for the construction of a stable genetic transformation system for peaches. This method forms the basis of peach molecular breeding and has broad application prospects in variety improvement.

[0018] This invention uses the cotyledons of mature peach seeds as explants. The sterilization method is simple and low-cost. It has the advantages of easy explant sourcing, no seasonal restrictions, year-round repeatability, and high regeneration efficiency. Attached Figure Description

[0019] Figure 1 This is a picture of the cotyledon explants of mature peach seeds in Example 1 during the culture period on adventitious bud induction medium.

[0020] Figure 2 Images showing the callus formation period induced on peach leaf explants in Example 1.

[0021] Figure 3 This is a picture of the adventitious bud development period on the peach leaf explant of Example 1.

[0022] Figure 4 This is a picture of the growth stage of peach adventitious buds in Example 1.

[0023] Figure 5 This is a picture of the adventitious root growth stage of peach in Example 1.

[0024] Figure 6 This is a picture of the complete regenerated plant during the soil cultivation period in Example 1.

[0025] Figure 7 The induction effect of cotyledonary explants from mature peach seeds on S1, S2, S3, S5, S6 and S7 culture media was investigated. Detailed Implementation

[0026] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0027] Example 1

[0028] (1) Culture medium preparation

[0029] First, the adventitious shoot induction medium was prepared as follows: MS + 4 mg / L 6-BA + 0.1 mg / L IBA + 30 g / L sucrose + 8 g / L agar, pH = 5.8. Second, the adventitious shoot elongation medium was prepared as follows: MS + 0.5 mg / L 6-BA + 0.1 mg / L IBA + 30 g / L sucrose + 8 g / L agar, pH = 5.8. Finally, the adventitious root induction medium was prepared as follows: MS + 2 mg / L 6-BA + 30 g / L sucrose + 8 g / L agar, pH = 5.8. All media were then autoclaved at 121°C for 20 minutes.

[0030] (2) Explant preparation

[0031] Select plump 'Zaoyoutao' peach pits, break open the outer shell to obtain peach seeds. Take mature peach seeds with intact, undamaged skin, soak them in a 3% hypochlorous acid solution for 30 minutes in a clean bench, rinse the seeds 6-8 times with sterile water, and then soak them for 12 hours, obtaining a total of 26 sterilized seeds. Place the sterilized and soaked seeds in a petri dish lined with filter paper and blot dry. Use a scalpel to cut off both ends of the seeds to remove the embryo, and further peel off the seed coat to obtain cotyledons with wounds at both ends as explants.

[0032] (3) Adventitious bud induction culture

[0033] Cotyledon explants were cultured on adventitious shoot induction medium (MS + 4 mg / L 6-BA + 0.1 mg / L IBA + 30 g / L sucrose + 8 g / L agar, pH = 5.8). Figure 1 ), inducing callus formation at the explant wound site ( Figure 2 ) and adventitious bud formation ( Figure 3 The cultivation conditions are: light intensity of 800-1200 Lux, light cycle of 16h light / 8h darkness, cultivation temperature of 24℃-26℃, and relative humidity of about 65%.

[0034] (4) Adventitious bud elongation growth culture

[0035] Once the adventitious buds have grown to 0.5-1 cm in length, use a scalpel to cut them off from the cotyledon explant (taking care to avoid including callus tissue). Then, transfer them to adventitious bud elongation medium (MS + 0.5 mg / L 6-BA + 0.1 mg / L IBA + 30 g / L sucrose + 8 g / L agar, pH = 5.8) for culture, allowing the adventitious buds to elongate and grow normally. Figure 4 To obtain well-growing adventitious buds, the cultivation conditions are: light intensity of 800-1200 Lux, light cycle of 16h light / 8h darkness, cultivation temperature of 24℃-26℃, and relative humidity of about 65%.

[0036] (5) Induction of adventitious roots

[0037] After the adventitious buds have grown to 3-5 cm in length, the base of the bud segment is thinly cut with a scalpel to create a wound, and then transferred to adventitious root induction medium (MS + 2 mg / L 6-BA + 30 g / L sucrose + 8 g / L agar, pH = 5.8) for culture to induce adventitious root development and growth. Figure 5 This process yielded well-growing tissue culture seedlings. The culture conditions were: light intensity of 800-1200 Lux, a photoperiod of 16 hours of light / 8 hours of darkness, a culture temperature of 24-26℃, and a relative humidity of approximately 65%.

[0038] (6) Seedling hardening and transplanting

[0039] Once the tissue culture seedlings have developed well-developed root systems, 3 mL of sterile water is poured into the culture bottle to cover the entire surface of the culture medium. The bottle cap is then opened, and the seedlings are hardened off in the tissue culture room. During hardening-off, care is taken to maintain the amount of sterile water in the bottle (ensuring that the sterile water completely submerges the roots of the seedlings). After 5 days of hardening-off, the tissue culture seedlings are removed, the culture medium is washed off the roots, and then they are transplanted into nutrient soil (prepared by mixing perlite and humus in a volume ratio of 1:2). The growth conditions in the nutrient soil are: light intensity of 1200-1600 Lux, photoperiod of 16 hours of light / 8 hours of darkness, greenhouse temperature of 22℃-24℃, and relative humidity of approximately 65%. This method yields healthy, fully regenerated peach seedlings through soil cultivation. Figure 6 ).

[0040] The regeneration efficiency of adventitious buds from cotyledon explants of mature 'Zao You Tao' seeds in step (3) of this embodiment was statistically analyzed, and the results are shown in Table 1.

[0041] Table 1. Adventitious bud regeneration efficiency of 'Zaoyoutao' cotyledon explants

[0042]

[0043] The adventitious bud induction medium and tissue culture method of this embodiment can induce the production of adventitious buds in peach leaf explants. Furthermore, through adventitious bud elongation culture and adventitious root induction, complete regenerated tissue culture seedlings can be obtained, laying a technical foundation for the construction of a peach genetic transformation system.

[0044] Comparative Example 1: Screening of Adventitious Bud Induction Culture Medium

[0045] When screening adventitious bud induction media, MS was used as the basal medium + 30 g / L sucrose + 8 g / L agar (pH = 5.8), and seven groups with different hormone ratios were set up (Table 2). All explants were cotyledon explants prepared using the method in Example 1. During the screening process, only S4 (MS + 4 mg / L 6-BA + 0.1 mg / L IBA + 30 g / L sucrose + 8 g / L agar, pH = 5.8), i.e., the adventitious bud induction medium in Example 1, could induce the production of normally developing adventitious buds from peach leaves. S1, S2, and S3 could only induce callus formation; S5 and S6 could only induce callus and adventitious bud-like tissues, which were highly susceptible to browning and necrosis and could not form normal adventitious buds; S7 induced adventitious root formation (…). Figure 7 ).

[0046] Table 2 Hormone Concentrations in Adventitious Bud Induction Culture Medium

[0047]

Claims

1. A tissue culture method for inducing adventitious shoot regeneration using peach leaf as explant, characterized by, It comprises the following steps: a. taking mature and intact peach seeds, disinfecting, absorbing surface moisture, cutting off both ends of the seeds to remove seed embryos, peeling the seed coat, and obtaining cotyledons with wounds at both ends as explants; the peach is of the 'Jiaoyoutao' variety; b. placing the cotyledon explants on adventitious bud induction medium to induce callus production and further induce adventitious bud occurrence; the adventitious bud induction medium is: MS + 4 mg / L 6-BA + 0.1 mg / L IBA + 30 g / L sucrose + 8 g / L agar, pH=5.8; c. after the adventitious buds grow to 0.5-1 cm long, cutting the adventitious buds from the cotyledon explants and moving them to adventitious bud elongation medium for culture to make the adventitious buds elongate and grow; the adventitious bud elongation medium is: MS + 0.5 mg / L 6-BA + 0.1 mg / L IBA + 30 g / L sucrose + 8 g / L agar, pH=5.8; d. after the adventitious buds grow to 3-5 cm long, cutting the adventitious buds from the bud segments at the base and moving them to adventitious root induction medium for culture to induce adventitious root occurrence and growth; the adventitious root induction medium is: MS + 2 mg / L 6-BA + 30 g / L sucrose + 8 g / L agar, pH=5.8; after growing into complete tissue culture seedlings, they are acclimatized in time; e. moving the acclimatized tissue culture seedlings to nutrient soil for culture; the culture conditions in steps b-d are: light intensity 800-1200 Lux, light cycle 16 h light / 8 h darkness, temperature 24℃-26℃, relative humidity 65%.

2. The method of claim 1, wherein, the disinfection is soaking the peach seeds in 3% mass fraction aqueous hypochlorous acid solution for 30 min, and then washing the seeds with sterile water for 6-8 times and soaking for 12 h.

3. The method of claim 1, wherein, the acclimatization is after the complete tissue culture seedlings with developed root systems are grown, 3 mL of sterile water is injected into the culture bottle to cover the entire culture medium surface, the bottle cap is opened, and the acclimatization is carried out in the tissue culture room; after 5 days of acclimatization, the tissue culture seedlings are taken out and the culture medium on the roots is washed clean.

4. The method of claim 1, wherein, the nutrient soil is prepared by mixing perlite: humus at a volume ratio of 1:

2.

5. The method of claim 1, wherein, the culture conditions in step e are: light intensity 1200-1600 Lux, light cycle 16 h light / 8 h darkness, temperature 22℃-24℃, relative humidity 65%.

Images