Tissue culture method for inducing regeneration of adventitious buds by taking peach leaves as explants

Abstract

The invention discloses a tissue culture method for inducing adventitious bud regeneration by taking peach leaves as explants. According to the method, the problem that the adventitious buds of peaches are difficult to regenerate is solved, the adventitious buds are successfully generated from cotyledons of mature seeds of peaches through induction of the adventitious bud induction culture medium, tissue culture seedlings of complete regenerated plants are formed through bud elongation culture and adventitious root induction, and seedlings which grow normally are further obtained through seedling hardening and domestication. According to the method, the cotyledons of the mature peach seeds are taken as the explants, the disinfection and sterilization method is simple and convenient, the cost is low, and the method has the advantages that the explants are easily available, are not limited by seasons, can be repeatedly carried out all year round and are high in regeneration efficiency; the method provides technical support for construction of a stable genetic transformation system of peaches, is a basis for molecular breeding of peaches, and has wide application prospects in variety improvement.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture method for inducing adventitious bud regeneration by taking peach leaves as explants. Background Art

[0002] Peach (Prunus persica) is native to China and is a perennial woody plant of the genus Prunus, subgenus Prunus, in the family Rosaceae. It is deeply loved by consumers for its high ornamental value, good fruit flavor and rich nutrition. Molecular breeding technology can significantly accelerate the selection and breeding process of high-quality peach varieties, thereby meeting the market's growing demand for quality traits. However, as a perennial woody fruit tree, the genetic transformation of peach has always been a recognized technical problem. At present, we have successfully established a stable genetic transformation system for peach roots and callus tissue, but the difficulty of adventitious bud regeneration is still a bottleneck restricting the establishment of a genetic transformation system for complete peach plants. Therefore, the establishment of an efficient adventitious bud regeneration system will provide important technical support for the construction of a stable genetic transformation system for peach, and then promote 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 the present invention is to provide a tissue culture method for inducing adventitious bud regeneration using peach leaves as explants. The culture medium and method of the present invention can achieve adventitious bud regeneration of peach leaf explants, and the material is simple to obtain, is not limited by time, and has high regeneration efficiency.

[0004] The present invention discloses 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, sterilize them, dry the surface moisture, cut off both ends of the seeds to remove the embryo, peel off the seed coat, and obtain the cotyledons with wounds on both ends as explants;

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

[0007] c. After the adventitious buds grow to 0.5-1 cm in length, the adventitious buds are cut from the cotyledon explants and transferred to the adventitious bud elongation medium for culture, so that 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;

[0008] d. After the adventitious buds grow to 3 - 5 cm in length, cut the adventitious buds from the base of the bud segment and transfer them to an adventitious root induction medium for culture 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 growing into complete tissue - cultured seedlings, acclimatize the seedlings in a timely manner;

[0009] e. Transplant the acclimatized tissue - cultured seedlings into nutrient soil for culture.

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

[0011] Preferably, the disinfection is to soak the peach seeds in a 3% (mass fraction) aqueous solution of hypochlorous acid for 30 min, wash the seeds 6 - 8 times with sterile water and then soak for 12 h.

[0012] Preferably, for the acclimatization, after growing into complete tissue - cultured seedlings with well - developed roots, inject 3 mL of sterile water into the culture bottle to cover the entire surface of the medium, open the bottle cap, acclimatize the seedlings in the tissue - culture room, take out the tissue - cultured seedlings after 5 days of acclimatization, and wash the medium on the roots clean.

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

[0014] Preferably, the culture conditions in steps b - d are: light intensity 800 - 1200 Lux, light cycle 16 h light / 8 h dark, temperature 24℃ - 26℃, relative humidity 65%.

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

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

[0017] During the induction of adventitious buds with the cotyledons of mature peach seeds as explants in the present invention, the formula of the adventitious bud induction medium and the selection of explants are the key; adventitious buds are successfully induced from the cotyledons of mature peach seeds using this adventitious bud induction medium, solving the problem of difficult adventitious bud regeneration in peaches. Through the elongation culture and rooting of adventitious buds, tissue - cultured seedlings that can be regenerated from scratch can be obtained, providing technical support for the construction of a stable genetic transformation system for peaches. The method of the present invention is the basis for peach molecular breeding and has broad application prospects in variety improvement.

[0018] The present invention uses the cotyledons of mature peach seeds as explants, and the disinfection and sterilization method is simple and low - cost. It has the advantages of easy explant collection, not being restricted by seasons, being repeatable throughout the year, and high regeneration efficiency. Brief Description of the Drawings

[0019] Figure 1 Picture of the cotyledon explants of the mature seeds of peach in Example 1 during the culture on the adventitious bud induction medium.

[0020] Figure 2 Picture of the period when callus is induced on the cotyledon explants of peach in Example 1.

[0021] Figure 3 Picture of the adventitious bud occurrence period on the cotyledon explants of peach in Example 1.

[0022] Figure 4 Picture of the adventitious bud growth period of peach in Example 1.

[0023] Figure 5 Picture of the adventitious root growth period of peach in Example 1.

[0024] Figure 6 Picture of the period of the complete regenerated plant in soil culture in Example 1.

[0025] Figure 7 Induction effects of the cotyledon explants of mature peach seeds on media S1, S2, S3, S5, S6 and S7. Detailed implementation manners

[0026] The following examples are further descriptions of the present invention rather than limitations thereof.

[0027] Example 1

[0028] (1) Preparation of media

[0029] First, prepare the adventitious bud induction medium, that is, MS + 4 mg / L 6 - BA + 0.1 mg / L IBA + 30 g / L sucrose + 8 g / L agar, pH = 5.8; secondly, prepare the adventitious bud elongation medium, that is, MS + 0.5 mg / L 6 - BA + 0.1 mg / L IBA + 30 g / L sucrose + 8 g / L agar, pH = 5.8; finally, prepare the adventitious root induction medium, that is, MS + 2 mg / L 6 - BA + 30 g / L sucrose + 8 g / L agar, pH = 5.8; autoclave at 121 °C for 20 min.

[0030] (2) Preparation of explants

[0031] Select peach pits of the 'Jujube Nectarine' variety with a plump appearance, break the shell of the pit, and obtain peach seeds. Take mature peach seeds with intact skin and no wounds, soak the peach seeds in a 3% hypochlorous acid aqueous solution in a clean bench for 30 minutes, wash the seeds with sterile water 6-8 times and soak them for 12 hours, and obtain a total of 26 sterilized seeds. Place the sterilized and soaked seeds in a culture dish with filter paper and absorb the surface moisture. Use a scalpel to cut off both ends of the seeds to remove the embryo, further peel off the seed coat, and obtain the cotyledons with wounds on both ends as explants.

[0032] (3) Adventitious bud induction culture

[0033] The cotyledon explants were placed on adventitious bud induction medium (MS + 4 mg / L 6-BA + 0.1 mg / L IBA + 30 g / L sucrose + 8 g / L agar, pH = 5.8) and cultured ( Figure 1 ), inducing callus formation at the wound of explants ( Figure 2 ) and adventitious budding ( Figure 3 The culture conditions are as follows: light intensity 800-1200 Lux, photoperiod 16 h light / 8 h dark, culture temperature 24° C.-26° C., and relative humidity about 65%.

[0034] (4) Adventitious bud elongation growth culture

[0035] When the adventitious buds grow to 0.5-1 cm in length, cut them off from the cotyledon explants with a scalpel (be careful not to leave callus tissue), and 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) to allow the adventitious buds to elongate normally ( Figure 4 ) to obtain adventitious buds with good growth. The culture conditions are light intensity 800-1200 Lux, light cycle 16h light / 8h dark, culture temperature 24℃-26℃, relative humidity about 65%.

[0036] (5) Adventitious root induction

[0037] When the adventitious buds grow to 3-5 cm in length, cut the base of the buds with a scalpel to make wounds, and then transfer them to adventitious root induction medium (MS + 2 mg / L 6-BA + 30 g / L sucrose + 8 g / L agar, pH = 5.8) to induce adventitious root formation and growth ( Figure 5 ), and obtain tissue culture seedlings with good growth. The culture conditions are light intensity 800-1200 Lux, photoperiod 16h light / 8h dark, culture temperature 24°C-26°C, and relative humidity about 65%.

[0038] (6) Hardening and transplanting

[0039] When the complete tissue culture seedlings with well-developed roots grow up, inject 3 mL of sterile water into the culture bottle to cover the entire surface of the medium, and open the bottle cap. Acclimatize the seedlings in the tissue culture room. During acclimatization, pay attention to maintaining the amount of sterile water in the bottle (keep the sterile water submerging the roots of the seedlings). After 5 days of acclimatization, take out the tissue culture seedlings, wash the medium on the roots, and transplant them into the 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, light cycle of 16 h light / 8 h darkness, greenhouse environmental temperature of 22°C - 24°C, and relative humidity of about 65%. Thus, healthy and growing complete regenerated peach seedlings are obtained by soil culture ( Figure 6 ).

[0040] The adventitious bud regeneration efficiency of the cotyledon explants of the 'Zaoyoutao' mature seeds in step (3) of this example was counted, and the results are shown in Table 1.

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

[0042]

[0043] Using the adventitious bud induction medium and tissue culture method of this example, adventitious buds can be induced from peach cotyledon explants. Further, complete regenerated tissue culture seedlings can be obtained through adventitious bud elongation culture and adventitious root induction, laying a technical foundation for the construction of the peach genetic transformation system.

[0044] Comparative Example 1: Screening of adventitious bud induction medium

[0045] When screening the adventitious bud induction medium, with MS as the basic medium + 30 g / L sucrose + 8 g / L agar (pH = 5.8), 7 groups of different hormone ratios were set (Table 2), and the explants were all cotyledon explants prepared by the method of 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), that is, the adventitious bud induction medium in Example 1, could induce the production of normally developed adventitious buds on peach cotyledons. S1, S2, and S3 could only induce the formation of callus; S5 and S6 could only induce the production of callus and adventitious bud-like tissues, and these tissues were extremely prone to browning and necrosis and could not form normal adventitious buds; S7 induced the occurrence of adventitious roots ( Figure 7 ).

[0046] Table 2 Hormone concentrations of adventitious bud induction medium

[0047]

Claims

1. A tissue culture method for inducing adventitious bud regeneration with peach leaves as explants, characterized in that, The following steps are involved: a. Take mature and intact peach seeds, sterilize them, dry the surface moisture, cut off both ends of the seeds to remove the embryo, peel off the seed coat, and obtain the cotyledons with wounds on both ends as explants; b. The cotyledon explants are placed on an adventitious bud induction medium to induce adventitious buds; the adventitious bud induction medium is: MS + 4mg / L 6-BA + 0.1mg / L IBA + 30g / L sucrose + 8g / L agar, pH = 5.8; c. After the adventitious buds grow to 0.5-1 cm in length, the adventitious buds are cut from the cotyledon explants and transferred to the adventitious bud elongation medium for culture, so that 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 in length, the adventitious buds are cut from the base of the bud segments and transferred to adventitious root induction medium for culture to induce adventitious root occurrence and growth; the adventitious root induction medium is: MS + 2mg / L 6-BA + 30g / L sucrose + 8g / L agar, pH = 5.8; after the complete tissue culture seedlings are grown, the seedlings are hardened in time; e. Transplant the tissue culture seedlings after hardening into nutrient soil for cultivation.

2. The method according to claim 1, characterized in that The peach is of the 'Zaoyounectarine' variety.

3. The method according to claim 1, wherein The disinfection is carried out by soaking peach seeds in a 3% hypochlorous acid aqueous solution for 30 minutes, washing the seeds with sterile water for 6-8 times and then soaking them for 12 hours.

4. The method according to claim 1, characterized in that The seedling hardening is to inject 3mL of sterile water into the culture bottle to cover the entire surface of the culture medium after the complete tissue culture seedlings with developed root systems are grown, open the bottle cap, and harden the seedlings in the tissue culture room. After hardening for 5 days, the tissue culture seedlings are taken out and the culture medium on the roots is washed clean.

5. The method according to claim 1, wherein The nutrient soil is prepared by mixing perlite and humus in a volume ratio of 1:

2.

6. The method according to claim 1, characterized in that, The culture conditions in steps bd are: light intensity 800-1200 Lux, light cycle 16 h light / 8 h dark, temperature 24° C.-26° C., and relative humidity 65%.

7. The method according to claim 1, characterized in that, The culture conditions in step e are: light intensity 1200-1600 Lux, light cycle 16 h light / 8 h dark, temperature 22° C.-24° C., and relative humidity 65%.

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