A method for rapidly and efficiently inducing differentiation of passion fruit callus into seedlings

Abstract

The present application relates to a kind of guava callus rapid and efficient induction differentiation seedling method.Taking the stem section of guava as explant, disinfection treatment is carried out;The explant after disinfection treatment is inoculated into callus induction medium and is induced to culture, and light yellow callus is obtained;Light yellow callus is inoculated into callus subculture and is subcultured, and the callus after subculture is obtained;The callus after subculture is inoculated into callus green conversion medium and is green conversion culture, and the callus after green conversion is obtained;The callus after green conversion is inoculated into callus differentiation medium and is differentiated to sprout, and the callus with sprout is obtained;The callus with sprout is inoculated into seedling culture medium and is seedling culture, and seedling is obtained;After seedling is divided into single seedling, it is inoculated into rooting culture medium and is rooted culture, and rooted seedling is obtained.The present application solves the technical bottleneck that guava callus is not easy to differentiate into bud, and can realize the rapid seedling of guava.

Description

Technical Field

[0001] This invention relates to a method for rapidly and efficiently inducing passion fruit callus to differentiate into seedlings, belonging to the field of biotechnology. Background Technology

[0002] Passion fruit, also known as granadilla, is a general term for fruits belonging to the genus Passiflora in the family Passifloraceae. Currently, there are over 500 species of Passiflora worldwide, of which about 60 have edible fruits. However, only 6 varieties are commercially cultivated for fresh consumption. In my country, two varieties are mainly cultivated: purple passion fruit and yellow passion fruit (commonly known as golden passion fruit). Suitable cultivation areas for passion fruit in my country are mainly distributed in Guangxi, Fujian, Guangdong, Yunnan, Guizhou, and Hainan. Currently, passion fruit seedlings on the market are mainly obtained through conventional cuttings and grafting from non-virus-free plants. Due to the lack of a mature tissue culture rapid propagation technology system for passion fruit, conventional cutting and grafting inevitably inherit the virus-carrying traits of the parent plant, causing widespread viral diseases in orchards and leading to industry problems such as weak seedling growth and deformed fruit.

[0003] The problem of passion fruit seedlings has become a bottleneck restricting the development of the passion fruit industry. Establishing a rapid propagation system for passion fruit tissue culture seedlings using plant tissue culture technology is of great and far-reaching significance for the detoxification of passion fruit plants, the preservation of superior traits, and biotechnology breeding, and will also have an important impact on the development of the passion fruit industry.

[0004] Numerous studies have been reported on the in vitro regeneration culture of passion fruit, mainly using different organ parts of passion fruit, such as internodes, axillary buds, leaves, ovaries, cotyledons, hypocotyls, tendrils, and endosperm, as explant materials to establish rapid propagation systems, forming regenerated plants from adventitious buds or clustered buds. However, there are few reports on the development of plants from callus differentiation, and these studies mainly focus on purple fruit, while rapid propagation techniques for inducing callus differentiation from yellow fruit into a large number of tissue culture seedlings are extremely rare. Summary of the Invention

[0005] The purpose of this invention is to solve the problems of difficult callus formation and differentiation into seedlings in lily in the prior art, and to provide a method for rapidly and efficiently inducing passion fruit callus to differentiate into seedlings.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a method for rapidly and efficiently inducing passion fruit callus to differentiate into seedlings. The method involves taking passion fruit stem segments as explants and sterilizing them; inoculating the sterilized explants into a callus induction medium for induction culture to obtain pale yellow callus; inoculating the pale yellow callus into a callus subculture medium for subculture to obtain subcultured callus; inoculating the subcultured callus into a callus greening medium for greening culture to obtain greened callus; inoculating the greened callus into a callus differentiation medium to differentiate and culture buds to obtain callus with buds; inoculating the callus with buds into a seedling culture medium for seedling culture to obtain basal seedlings; and dividing the basal seedlings into individual seedlings and inoculating them into a rooting medium for rooting culture to obtain rooted seedlings. The formulations for the callus induction medium were as follows: MS + 0.6 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 3 g / L plant gel, pH = 5.5–5.8; the formulation for callus subculture was: MS + 0.6 mg / L 6-BA + 0.06 mg / L IAA + 30 g / L sucrose + 3 g / L plant gel, pH = 5.5–5.8; the formulation for callus greening medium was: MS + 0.2 mg / L 6-BA + 0.2 mg / L IAA + 30 g / L sucrose + 3 g / L plant gel, pH = 5.5–5.8; the formulation for callus differentiation medium was: MS + 0.4 mg / L 6-BA + 0.04 mg / L IAA + 30 g / L sucrose + 3 g / L plant gel, pH = 5.5–5.8; and the formulation for seedling culture medium was: MS + 0.6 mg / L 6-BA + 0.04 mg / L IAA + 30 g / L sucrose + 3 g / L plant gel, pH = 5.5–5.8. The rooting medium formula is: 1 / 2 MS + 0.4 mg / L 6-BA + 0.2 mg / L IBA + 30 g / L sucrose + 15 g / L plant gel, pH = 5.5–5.8; The disinfection process is as follows: first disinfect with 75% alcohol for 1-2 minutes, then rinse with sterile water 4-5 times, then disinfect with 1% mercuric chloride solution for 10 minutes, then rinse with sterile water 5-7 times, and air dry naturally. The induction culture conditions were as follows: cultured for 32 days at a temperature of 27℃, a light intensity of 3000 lux, and a daily light duration of 16h. The subculture conditions are as follows: subculture 2 to 4 times under the conditions of 27℃ temperature, 3000 lux light intensity and 16h light time per day, with a subculture cycle of 7 days / time. The conditions for greening culture are: cultured for 7 to 10 days at a temperature of 27℃, a light intensity of 3000 lux, and a daily light duration of 16 hours. The differentiation culture conditions were as follows: cultured for 7 to 14 days at a temperature of 27℃, a light intensity of 3000 lux, and a daily light duration of 16 hours. The conditions for seedling cultivation are as follows: cultivation for 7 to 15 days under the conditions of 27℃ temperature, 3000 lux light intensity, and 16h light duration per day; The conditions for rooting culture are as follows: culture for 7 to 15 days at a temperature of 27℃, a light intensity of 3000 lux, and a daily light duration of 16 hours.

[0007] The second aspect of the present invention provides the application of the above-mentioned method for rapid and efficient induction of passion fruit callus into seedlings in passion fruit propagation.

[0008] The significant advantages of this invention are: This invention has yielded an important callus material from passion fruit, specifically greening callus tissue. For the first time, it has produced a callus material from passion fruit that can be rapidly induced to differentiate into seedlings, thus improving the efficiency of callus differentiation. Secondly, it enables the establishment of a system for inducing seedling differentiation from callus tissue in a relatively short time, representing a breakthrough in the efficient method of inducing seedlings from lily callus tissue. This significantly enhances the ability of passion fruit tissue culture to rapidly propagate seedlings, providing an important technological platform for the future industrial-scale propagation of passion fruit tissue culture seedlings. Attached Figure Description

[0009] Figure 1 The flowchart of the method for rapidly and efficiently inducing passion fruit callus to differentiate into seedlings according to the present invention is as follows: A. Pale yellow callus; B. Subcultured callus; C. Callus after turning green; D. Callus with buds; E. Seedling; F. Rooted seedling. Detailed Implementation

[0010] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0011] Example 1: This embodiment provides a method for rapidly and efficiently inducing passion fruit callus tissue to differentiate into seedlings, which is carried out according to the following steps: S1: Collect stem segments of passion fruit yellow fruit "Qinmi No. 9" with a length of 4-6cm as explants and store them in a refrigerator at 4℃ for later use.

[0012] S2: Take out the passion fruit stem segments stored in the 4℃ refrigerator and disinfect them. The disinfection process is as follows: first disinfect with 75% (v / v) alcohol for 1-2 minutes, then rinse with sterile water 4-5 times for 2 minutes each time, then disinfect with 1% (w / v) mercuric chloride solution for 10 minutes, and finally rinse with sterile water 5-7 times for 2 minutes each time, and let it air dry naturally.

[0013] S3: Sterilized passion fruit stem segments were cut into lengths of 2-3 cm and inoculated into callus induction medium (MS + 0.6 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 3 g / L plant gel, pH = 5.5-5.8). The cells were cultured at 27℃, light intensity of 3000 lux, and daily photoperiod of 16 h. After 32 days, pale yellow callus tissue formed on the stem segments; the callus induction rate reached over 75%.

[0014] S4: The pale yellow callus tissue was cut into small pieces of 2-5 mm and inoculated into callus tissue subculture medium (MS + 0.6 mg / L 6-BA + 0.06 mg / L IAA + 30 g / L sucrose + 3 g / L plant gel, pH = 5.5-5.8). It was subcultured 2-4 times under the conditions of 27℃, light intensity 3000 lux, and daily photoperiod 16 h, with a subculture cycle of 7 days / time, to obtain subcultured callus tissue; the callus tissue growth index reached 51%, and the browning rate was only 2%.

[0015] S5: Callus tissue that has been subcultured more than 3 times was inoculated into callus greening medium (MS + 0.2 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 3 g / L plant gel, pH = 5.5-5.8) and cultured at 27℃, light intensity 3000 lux, and daily photoperiod 16 h. After 7-10 days, the pale yellow callus tissue gradually turned green, and the greened callus tissue was obtained; the greening rate of the callus tissue reached 78%.

[0016] S6: The greened callus tissue was inoculated into callus differentiation medium (MS + 0.4 mg / L 6-BA + 0.04 mg / L IIA + 30 g / L sucrose + 3 g / L plant gel, pH = 5.5-5.8) and cultured at 27℃, light intensity 3000 lux, and daily photoperiod 16 h. After 7-14 days, buds formed on the callus tissue, and callus tissue with buds was obtained; the callus differentiation rate reached more than 85%.

[0017] S7: Callus tissue with buds was inoculated into seedling culture medium (MS + 0.6 mg / L 6-BA + 0.06 mg / L IIA + 30 g / L sucrose + 3 g / L plant gel, pH = 5.5-5.8) and cultured at 27℃, light intensity 3000 lux, and daily photoperiod 16 h. After 7-15 days, buds on the callus tissue gradually formed clusters of seedlings; the differentiation rate of buds into clusters of seedlings reached over 85%.

[0018] S8: After separating the clump seedlings from the callus base into individual seedlings, they were inoculated into rooting medium (1 / 2 MS + 0.4 mg / L 6-BA + 0.2 mg / L IBA + 30 g / L sucrose + 15 g / L plant gel, pH = 5.5-5.8) and cultured at 27℃, light intensity 3000 lux, and daily photoperiod 16 h. Rooting was completed in 7-15 days, and rooted seedlings were obtained; the rooting rate was over 90%, and the number of roots per seedling was 2-3.

[0019] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for rapidly and efficiently inducing passion fruit callus to differentiate into seedlings, characterized in that: Passion fruit stem segments were used as explants and sterilized. The sterilized explants were inoculated into callus induction medium for induction culture to obtain pale yellow callus. The pale yellow callus was then inoculated into callus subculture medium for subculture to obtain subcultured callus. The subcultured callus was inoculated into callus greening medium for greening culture to obtain greened callus. The greened callus was inoculated into callus differentiation medium for differentiation culture to obtain buds, resulting in callus with buds. The callus with buds was inoculated into seedling culture medium for seedling culture to obtain basal seedlings. The basal seedlings were divided into individual seedlings and inoculated into rooting medium for rooting culture to obtain rooted seedlings. The formulations for callus induction medium are: MS + 0.6 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 3 g / L plant gel, pH = 5.5~5.8; the formulation for callus subculture is: MS + 0.6 mg / L 6-BA + 0.06 mg / L IAA + 30 g / L sucrose + 3 g / L plant gel, pH = 5.5~5.8; the formulation for callus greening medium is: MS + 0.2 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 3 g / L plant gel, pH = 5.5~5.8; the formulation for callus differentiation medium is: MS + 0.4 mg / L 6-BA + 0.04 mg / L IAA + 30 g / L sucrose + 3 g / L plant gel, pH = 5.5~5.8; and the formulation for seedling culture medium is: MS + 0.6 mg / L 6-BA + 0.04 mg / L NAA + 30 g / L sucrose + 3 g / L plant gel, pH = 5.5~5.

8. The rooting medium formula is: 1 / 2 MS + 0.4 mg / L 6-BA + 0.2 mg / L IBA + 30 g / L sucrose + 15 g / L plant gel, pH=5.5~5.

8.

2. The method according to claim 1, characterized in that: The disinfection process is as follows: first disinfect with 75% alcohol for 1-2 minutes, then rinse with sterile water 4-5 times, then disinfect with 1% mercuric chloride solution for 10 minutes, then rinse with sterile water 5-7 times, and air dry naturally.

3. The method according to claim 1, characterized in that: The induction culture conditions were: 32 days of culture at a temperature of 27℃, a light intensity of 3000 lux, and a daily light duration of 16h.

4. The method according to claim 1, characterized in that: The conditions for subculture are as follows: subculture 2-4 times under the conditions of 27℃ temperature, 3000 lux light intensity and 16h light time per day, with a subculture cycle of 7 days / time.

5. The method according to claim 1, characterized in that: The conditions for greening culture are: culture for 7-10 days at a temperature of 27℃, a light intensity of 3000 lux, and a daily light duration of 16h.

6. The method according to claim 1, characterized in that: The conditions for differentiation culture were: 7-14 days of culture at a temperature of 27℃, a light intensity of 3000 lux, and a daily light duration of 16h.

7. The method according to claim 1, characterized in that: The conditions for seedling cultivation are: 7-15 days of cultivation at a temperature of 27℃, a light intensity of 3000 lux, and a daily light duration of 16 hours.

8. The method according to claim 1, characterized in that: The conditions for rooting culture are: 7-15 days of culture at a temperature of 27℃, a light intensity of 3000 lux, and a daily light duration of 16 hours.

9. The application of the method as described in claim 1 in passion fruit propagation.

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