Method for rapid and efficient induced differentiation seedling formation of passion fruit calluses

Abstract

The invention relates to a rapid and efficient induced differentiation seedling formation method for passion fruit calluses. The method comprises the following steps: taking stems of passion fruits as explants, and performing disinfection treatment; inoculating the disinfected explant into a callus induction culture medium for induction culture to obtain a faint yellow callus; inoculating the faint yellow callus into callus subculture for subculture, so as to obtain subcultured callus; inoculating the subcultured calluses into a callus greening culture medium for greening culture, so as to obtain the greened calluses; inoculating the greened calluses into a callus differentiation culture medium, and carrying out differentiation culture to obtain buds, so as to obtain the calluses with the buds; inoculating the calluses with the buds into a seedling culture medium for seedling culture, so as to obtain subordinate seedlings; dividing the subordinate seedlings into single seedlings, inoculating the single seedlings into a rooting culture medium, and performing rooting culture to obtain rooted seedlings. According to the method, the technical bottleneck that the passion fruit calluses are not easy to differentiate into buds is solved, and rapid seedling culture of the passion fruits can be realized.

Description

Technical Field

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

[0002] Passion fruit, also known as passion fruit, is the general name for fruits of the genus Passiflora in the family Passifloraceae. There are more than 500 species of passion fruit plants in the world, of which about 60 species have edible fruits. However, there are only 6 species of fresh food that can actually be used for commercial cultivation. There are two main varieties cultivated in my country: purple passion fruit and yellow passion fruit (commonly known as golden passion fruit). The areas suitable for cultivating passion fruit in my country are mainly distributed in Guangxi, Fujian, Guangdong, Yunnan, Guizhou and Hainan. At present, passion fruit seedlings on the market are mainly obtained from conventional cuttings and grafted seedlings with non-virus-free plants as the mother parent. Due to the lack of a mature passion fruit tissue culture rapid propagation technology system, conventional cutting seedlings and grafted seedlings inevitably inherit the toxic characteristics of the mother parent, causing the virus disease to spread over a large area in the orchard, leading to industrial problems such as weak seedling growth and fruit deformity.

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

[0004] There have been many reports 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 a rapid propagation system, forming regenerated plants with adventitious buds or clustered buds, while there are few reports on the formation of plants by callus differentiation, and most of them are purple fruits, while there are very few rapid propagation techniques for inducing callus tissue of yellow fruits to differentiate into a large number of tissue culture seedlings. Summary of the invention

[0005] The invention aims to solve the problems of difficulty in forming lily callus and differentiation into seedlings 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 purpose, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a method for rapidly and efficiently inducing passion fruit callus to differentiate into seedlings, which comprises the following steps: taking a passion fruit stem segment as an explant and performing a disinfection treatment; inoculating the disinfected explant into a callus induction medium for induction culture to obtain a light yellow callus; inoculating the light yellow callus into a callus subculture medium for subculture culture to obtain a subcultured callus; inoculating the subcultured callus into a callus greening medium for greening culture to obtain a greening callus; inoculating the greening callus into a callus differentiation medium for differentiation culture to produce a bud to obtain a callus with a bud; inoculating the callus with a bud into a seedling culture medium for seedling culture to obtain a seedling; dividing the seedling into individual seedlings and then inoculating into a rooting medium for rooting culture to obtain a rooted seedling; Among them, the formula of callus induction medium is: MS+0.6mg / L 6-BA+0.2mg / LNAA+30g / L sucrose+3g / L plant gel, pH=5.5-5.8; the formula of callus subculture is: MS+0.6mg / L 6-BA+0.06mg / LIAA+30g / L sucrose+3g / L plant gel, pH=5.5-5.8; the formula of callus greening medium is: MS+0.2mg / L6-BA+0.2mg / L NAA+30g / L sucrose+3g / L plant gel, pH=5.5-5.8; the formula of callus differentiation medium is: MS+0.4mg / L 6-BA+0.04mg / L IAA+30g / L sucrose+3g / L plant gel, pH=5.5-5.8; the formula of seedling medium is: MS+0.6mg / L 6-BA+0.06mg / L IAA+30g / L sucrose+3g / L plant gel, pH=5.5-5.8; the formula of rooting medium is: 1 / 2MS+0.4mg / L 6-BA+0.2mg / L IBA+30g / L sucrose+15g / L plant gel, pH=5.5-5.8; The disinfection process is as follows: first disinfect with 75% alcohol for 1 to 2 minutes, then rinse with sterile water for 4 to 5 times, then disinfect with 1% mercuric chloride solution for 10 minutes, then rinse with sterile water for 5 to 7 times, and dry naturally; The induction culture conditions are: 32 days of culture at 27°C, light intensity 3000 lux, and single-day light time 16h; The conditions for subculture are: subculture 2 to 4 times at a temperature of 27°C, a light intensity of 3000 lux, and a single-day light duration of 16 hours, with a subculture cycle of 7 days / time; The greening culture conditions are: 7 to 10 days at a temperature of 27°C, a light intensity of 3000 lux, and a single-day light duration of 16 hours; The conditions for differentiation culture are: culture at a temperature of 27°C, a light intensity of 3000 lux, and a single-day light duration of 16 hours for 7 to 14 days; The conditions for seedling cultivation are: 7 to 15 days at a temperature of 27°C, a light intensity of 3000 lux, and a single-day light duration of 16 hours; The rooting culture conditions are as follows: culture at a temperature of 27°C, a light intensity of 3000 lux, and a single-day light duration of 16 hours for 7 to 15 days.

[0007] The second aspect of the present invention provides the application of the above-mentioned method for rapidly and efficiently inducing differentiation of passion fruit callus into seedlings in passion fruit breeding.

[0008] The significant advantages of the present invention are: The present invention obtains the important callus material of passion fruit greening callus, and obtains the callus material that can be quickly induced and differentiated into seedlings on passion fruit for the first time, thereby improving the efficiency of callus induction and differentiation. Secondly, a system for inducing callus differentiation into seedlings can be established in a relatively short period of time, and a breakthrough method for inducing lily callus differentiation into efficient seedlings is achieved, which greatly improves the ability of passion fruit tissue to quickly propagate seedlings, and provides an important technical platform for the application of passion fruit tissue culture seedlings in industrialized breeding in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 : Flow chart of the method for rapidly and efficiently inducing passion fruit callus to differentiate into seedlings. A. Light yellow callus; B. Subcultured callus; C. Callus after turning green; D. Callus with buds; E. Seedlings from seedlings; F. Rooted seedlings. DETAILED DESCRIPTION

[0010] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0011] Embodiment 1: This embodiment provides a method for rapidly and efficiently inducing passion fruit callus to differentiate into seedlings, which is carried out according to the following steps: S1: Stem segments of passion fruit yellow fruit “Qinmi 9” with a length of 4 to 6 cm were collected as explants and stored in a 4°C refrigerator for later use.

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

[0013] S3: Cut the sterilized passion fruit stem segments into 2-3 cm in length, inoculate them into callus induction medium (MS+0.6 mg / L 6-BA+0.2 mg / L NAA+30 g / L sucrose+3 g / L phytogel, pH=5.5-5.8), and culture them under the conditions of temperature 27°C, light intensity 3000 lux, and single-day light time 16 h. After 32 days, light yellow callus is formed on the stem segments; the callus induction rate is over 75%.

[0014] S4: Cut the light yellow callus into small pieces of 2-5 mm, inoculate into callus subculture medium (MS+0.6 mg / L 6-BA+0.06 mg / L IAA+30 g / L sucrose+3 g / L phytogel, pH=5.5-5.8), subculture 2-4 times under the conditions of temperature 27°C, light intensity 3000 lux, and single-day light time 16 h, with a subculture cycle of 7 days / time, to obtain subcultured callus; the callus growth index reached 51%, and the browning rate was only 2%.

[0015] S5: The callus tissue that has been subcultured more than 3 times was inoculated into callus tissue 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 under the conditions of temperature 27 ° C, light intensity 3000 lux, and single-day lighting time 16 hours. After 7 to 10 days, the light yellow callus tissue gradually turned green to obtain the green callus tissue; the callus tissue greening rate reached 78%.

[0016] S6: The green callus was inoculated into callus differentiation medium (MS+0.4mg / L 6-BA+0.04mg / LIAA+30g / L sucrose+3g / L plant gel, pH=5.5-5.8), and cultured under the conditions of temperature of 27°C, light intensity of 3000lux, and single-day light time of 16h. After 7-14 days, buds were formed on the callus to obtain callus with buds; the callus differentiation rate was over 85%.

[0017] S7: The callus with buds was inoculated into a seedling medium (MS+0.6mg / L 6-BA+0.06mg / LIAA+30g / L sucrose+3g / L plant gel, pH=5.5-5.8), and cultured under the conditions of a temperature of 27°C, a light intensity of 3000lux, and a single-day lighting time of 16h. After 7-15 days, the buds on the callus gradually formed clustered seedlings; the differentiation rate of buds into clustered seedlings was more than 85%.

[0018] S8: The clustered seedlings are separated from the callus tissue base into individual seedlings and inoculated into rooting medium (1 / 2MS+0.4mg / L6-BA+0.2mg / L IBA+30g / L sucrose+15g / L plant gel, pH=5.5-5.8), and cultured under the conditions of temperature of 27°C, light intensity of 3000lux, and single-day light time of 16h. Rooting is completed after 7-15 days to obtain rooted seedlings; the rooting rate is more than 90%, and the number of roots of a single seedling is 2-3.

[0019] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within 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: The passion fruit stem segment is taken as an explant and disinfected; the disinfected explant is inoculated into a callus induction medium for induction culture to obtain light yellow callus; the light yellow callus is inoculated into a callus subculture medium for subculture culture to obtain subculture callus; the subculture callus is inoculated into a callus greening medium for greening culture to obtain greening callus; the greening callus is inoculated into a callus differentiation medium for differentiation culture to produce buds to obtain callus with buds; the callus with buds is inoculated into a seedling culture medium for seedling culture to obtain seedlings; the seedlings are divided into individual seedlings and inoculated into a rooting medium for rooting culture to obtain rooted seedlings.

2. The method according to claim 1, characterized in that: The formula of callus induction medium is: MS+0.6mg / L 6-BA+0.2mg / L NAA+30g / L sucrose+3g / L plant gel, pH=5.5~5.8; the formula of callus subculture is: MS+0.6mg / L 6-BA+0.06mg / L IAA+30g / L sucrose+3g / L plant gel, pH=5.5~5.8; the formula of callus greening medium is: MS+0.2mg / L 6-BA+0.2mg / L NAA+30g / L sucrose+3g / L plant gel, pH=5.5~5.8; the formula of callus differentiation medium is: MS+0.4mg / L 6-BA+0.04mg / L IAA+30g / L sucrose+3g / L plant gel, pH=5.5~5.8; the formula of seedling medium is: MS+0.6mg / L 6-BA+0.06mg / L IAA+30g / L sucrose+3g / L plant gel, pH=5.5~5.8; the formula of rooting medium is: 1 / 2 MS+0.4mg / L 6-BA+0.2mg / L IBA+30g / L sucrose+15g / L plant gel, pH=5.5~5.

8.

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

4. The method according to claim 1, characterized in that: The induction culture conditions are: culture for 32 days at a temperature of 27°C, a light intensity of 3000 lux, and a single-day light duration of 16 hours.

5. The method according to claim 1, characterized in that: The conditions for subculture are: subculture 2 to 4 times at a temperature of 27°C, a light intensity of 3000 lux, and a single-day light time of 16 hours, with a subculture cycle of 7 days / time.

6. The method according to claim 1, characterized in that: The conditions for greening culture are: culture for 7 to 10 days at a temperature of 27°C, a light intensity of 3000 lux, and a single-day lighting time of 16 hours.

7. The method according to claim 1, characterized in that: The conditions for differentiation culture are: culture at a temperature of 27°C, a light intensity of 3000 lux, and a single-day lighting time of 16 hours for 7 to 14 days.

8. The method according to claim 1, characterized in that: The conditions for seedling cultivation are: culture for 7 to 15 days at a temperature of 27°C, a light intensity of 3000 lux, and a single-day lighting time of 16 hours.

9. The method according to claim 1, characterized in that: The rooting culture conditions are: culture at a temperature of 27°C, a light intensity of 3000 lux, and a single-day light time of 16 hours for 7 to 15 days.

10. Application of the method as claimed in claim 1 in passion fruit breeding.

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