CN112106662B

CN112106662B - Passion fruit in-vitro rapid propagation and genetic transformation method

Info

Publication number: CN112106662B
Application number: CN202011106774.4A
Authority: CN
Inventors: 何锐杰; 曾黎辉; 余伟军
Original assignee: Fujian Agriculture and Forestry University
Current assignee: Fujian Agriculture and Forestry University
Priority date: 2020-10-16
Filing date: 2020-10-16
Publication date: 2022-12-23
Anticipated expiration: 2040-10-16
Also published as: CN112106662A

Abstract

The invention provides an in vitro rapid propagation and genetic transformation method for passion fruit, which comprises a large amount of rapid acquisition and in vitro rapid propagation methods for sterile explants and a genetic transformation method. The invention adopts the stem section of the aseptic seedling as the explant, effectively improves the phenomenon that a large amount of explants are browned and die in the genetic transformation of passion fruit, wherein the use of the low-concentration plant growth regulator of the in vitro rapid propagation culture medium can not only lead the plant to grow healthily and efficiently, but also reduce the cost in production.

Description

Passion fruit in-vitro rapid propagation and genetic transformation method

Technical Field

The invention relates to the field of horticultural plant propagation and new variety cultivation, in particular to an in vitro rapid propagation and genetic transformation method for passion fruit.

Background

The passion fruit propagation method mainly adopts cuttage propagation of seedlings and tendrils, and the tissue culture is less applied to production. The existing method for obtaining the in-vitro rapid propagation sterile explant of the passion fruit mainly depends on aseptic seeding of seeds or direct disinfection of field plants. The passion fruit tissue culture mainly adopts a general culture medium of an MS culture medium and 3 percent of cane sugar, and biological growth regulators with different proportions are respectively added in the steps of callus induction, cluster bud induction and the like, such as a callus induction culture medium: MS + 1.0 mg/L6-BA +0.2 mg/L NAA + 0.05 mg/L TDZ, cluster bud Induction Medium: MS + 1.0 mg/L6-BA +0.5 mg/L NAA + 0.05 mg/L TDZ. In seedling propagation, the seed germination rate and survival rate are low, the seedling recovering period is long, and the disease resistance is low; if the cuttage propagation of the vine strips is not operated properly, the degeneration of varieties and the increase of diseases are accelerated, so that the yield is reduced. In the process of obtaining the sterile explant, because passion fruit contains rich endophytes, the passion fruit cannot be effectively removed when in-vitro disinfection is carried out by using field passion fruit materials, and the explant is easy to die quickly even if disinfected by using high-concentration disinfectant for a long time; meanwhile, because the passion fruit seeds contain very thick seed shells and germinate under aseptic conditions, the germination rate is very low, and the embryonic axis is very easily damaged and cannot germinate even if the passion fruit seeds are treated in a physical shell breaking mode.

The existing passion fruit in-vitro rapid propagation technology does not screen the optimal sucrose concentration of a culture medium. Part of the schemes use plant growth regulators with relatively high concentration, abnormal growth phenomena such as internode shortening, wound callus expansion, lignification and the like can be found after actual operation, and an optimum passion fruit strong seedling culture medium is not screened. Meanwhile, cotyledons or hypocotyls are used as infection materials in the conventional genetic transformation, so that the acquisition cost is high, the efficiency is low, and particularly, the cotyledons are easy to die due to agrobacterium toxicity after infection. In addition, in the prior art, after infection is finished, co-culture is directly transferred to screening culture, and explants are easy to brown and die due to sudden contact with high-concentration antibiotics.

Disclosure of Invention

The invention aims to provide a method for in vitro rapid propagation and genetic transformation of passion fruit.

In order to realize the purpose, the invention adopts the following technical scheme:

an in vitro rapid propagation and genetic transformation method for passion fruit comprises the following steps:

(1) Rapid bulk harvest of sterile explants: peeling seeds from fruits, cleaning, soaking the seeds for 72h, planting the seeds in a 128-hole tray one by one hole, and taking autoclaved pure vermiculite as a substrate; placing the plug disc in an environment with the temperature of 28-32 ℃, and germinating the seeds 15 days later; cutting off the embryonic axis to the stem tip part when the cotyledon is just unfolded, washing the cotyledon until the cotyledon is clean, soaking the cotyledon in saturated washing powder water for 20min, then washing the cotyledon with running water for 30min, and finishing the subsequent steps in a superclean workbench; soaking the material in 75% w/v alcohol for 30s, washing with sterile double distilled water for 2 times, soaking with 7.5% w/v sodium hypochlorite solution for 10min, shaking the bottle body continuously, washing with sterile double distilled water for 3-6 times, and inoculating the explant into a growth culture medium for growth; after 30 days, the bud grows up, and the internode stem segment of the aseptic seedling is cut out to be used as a fast propagation explant;

(2) The in vitro rapid propagation method comprises the following steps: inoculating the fast propagation explant into a callus induction culture medium for culturing for 7 days, then transferring the callus induction culture medium into a differentiation culture medium for inducing cluster buds, cutting off the cluster buds, transferring the cut cluster buds into a rooting and seedling strengthening culture medium for culturing to obtain a tissue culture seedling; opening a cap of a tissue culture seedling growing strongly 2 days before transplanting for hardening the seedling, washing a residual culture medium at the root with tap water before transplanting, and then transplanting the seedling into a mixed matrix with the mass ratio of peat soil to perlite being 7: 3 for culturing to obtain an aseptic seedling;

(3) The genetic transformation method comprises the following steps: taking the stem section of the aseptic seedling in the step (2) as an infection material, pre-culturing the infection material in a callus induction culture medium for 3 days, soaking the infection material in an agrobacterium infection solution for 15min during infection, and continuously shaking the bottle body during infection; then transferring the callus to a callus induction culture medium without any antibiotic for co-culture for 3 days, transferring the callus to a callus induction culture medium containing 300mg/L carbenicillin for delayed culture for 7 days, then transferring the callus to a differentiation culture medium containing 50mg/L kanamycin and 300mg/L carbenicillin for differentiating cluster buds and carrying out screening culture to obtain a positive explant, and finally transferring the positive explant to a rooting and seedling strengthening culture medium containing 50mg/L kanamycin and 300mg/L carbenicillin for culture.

The preparation method of the agrobacterium infection solution comprises the following steps: selecting a single agrobacterium colony, placing the single agrobacterium colony in a 1.5ml centrifuge tube filled with 1ml YEB liquid culture medium, placing the centrifuge tube in a shaking table at 28 ℃, culturing for 8 hours at 200 rpm, transferring all bacteria liquid to a 100ml triangular flask filled with 50ml YEB liquid culture medium containing 200mg/L AS in an aseptic environment, placing the triangular flask in the shaking table at 28 ℃, culturing at 200 rpm until the od value of agrobacterium is =0.8, and sealing the triangular flask by using a ventilating sealing film in the agrobacterium culture process. The bacterial solution was centrifuged in a sterilized centrifuge tube at 5000rpm for 5min, the supernatant was decanted, resuspended to od =0.6 using 1/2MS liquid medium containing 3% sucrose +200mg/LAS, and then allowed to stand at room temperature for 3h.

The growth medium is MSM + 0.25mg/L TDZ + 0.1 mg/L IBA + 2wt.% sucrose; the callus induction culture medium MSM + 0.25mg/L TDZ +0.5 mg/L IBA + 2wt.% sucrose; the differentiation medium is MSM +0.5 mg/L TDZ + 0.1 mg/L IBA + 2wt.% sucrose; the rooting and seedling strengthening culture medium is MSM + 2wt.% of sucrose.

The invention has the advantages that:

the invention adopts the sterilization of the passion fruit seedlings germinating in the sterilized vermiculite to obtain aseptic seedlings, solves the problems that the aseptic explants can not be obtained directly from field plants because of the rich endophyte, and the aseptic seeds are utilized to sow the low germination rate in the sealed tissue culture bottle, and can quickly obtain a large amount of aseptic seedlings.

The invention adopts a low-concentration hormone proportion culture medium suitable for callus induction, cluster bud induction and plant rooting and seedling strengthening, and particularly, the sucrose content in the culture medium has obvious influence on the growth of explants.

The invention adopts the stem section of the aseptic seedling as the explant, and effectively improves the phenomenon that a large amount of explants are browned and die in the genetic transformation of passion fruit. The use of the low-concentration plant growth regulator of the in vitro rapid propagation culture medium can not only ensure that plants grow healthily and efficiently, but also reduce the cost in production.

Drawings

FIG. 1 Effect of sugar content in the medium on the growth of the cultured seedlings.

FIG. 2 high concentrations of IBA lead to extensive lignification of calli.

Detailed Description

Example 1

A method for in vitro rapid propagation and genetic transformation of passion fruit comprises the following steps:

(1) Large and rapid acquisition of sterile explants: peeling seeds from fruits, cleaning, soaking the seeds for 72h, planting the seeds in a 128-hole tray one by one hole, and taking autoclaved pure vermiculite as a substrate; placing the plug in an environment at 30 ℃ and germinating 15 days later; cutting off the embryonic axis to the stem tip part when the cotyledon is just unfolded, washing the cotyledon until the cotyledon is clean, soaking the cotyledon in saturated washing powder water for 20min, then washing the cotyledon with running water for 30min, and finishing the subsequent steps in a superclean workbench; soaking the material in 75% w/v alcohol for 30s, washing with sterile double distilled water for 2 times, soaking with 7.5% w/v sodium hypochlorite solution for 10min, shaking the bottle body continuously, washing with sterile double distilled water for 4 times, and inoculating the explant into a growth medium for growth; after 30 days, the bud seedlings grow up, and the internode stems of the aseptic seedlings are cut out to be used as fast propagation explants; the survival rate of the sterile explant reaches more than 99 percent. The survival rate of aseptic seedlings obtained by sterilizing passion fruit explants obtained from the field is very low, endophyte pollution can be avoided only when the concentration of sodium hypochlorite reaches 10%, but the explants are easy to brown or whiten along with the increase of the sterilization time. The survival rate of the aseptic seedlings can reach 6.67 percent only when the disinfection time is between 7.5 and 10 minutes.

(2) The in vitro rapid propagation method comprises the following steps: inoculating the fast propagation explant into a callus induction culture medium for culturing for 7 days, then transferring the explant into a differentiation culture medium for inducing cluster buds, wherein the proliferation coefficient can reach 22, cutting the cluster buds, transferring the cut cluster buds into a rooting and seedling strengthening culture medium for culturing to obtain a tissue culture seedling; opening a cap of a tissue culture seedling growing strongly 2 days before transplanting for hardening the seedling, washing a residual culture medium at the root with tap water before transplanting, and then transplanting the seedling into a mixed matrix with the mass ratio of peat soil to perlite being 7: 3 for culturing to obtain an aseptic seedling;

(3) The genetic transformation method comprises the following steps: taking the stem section of the aseptic seedling in the step (2) as an infection material, pre-culturing the infection material in a callus induction culture medium for 3 days, soaking the infection material in an agrobacterium infection solution for 15min during infection, and continuously shaking the bottle body during infection; then transferring the callus to a callus induction culture medium without any antibiotic for co-culture for 3 days, transferring the callus to a callus induction culture medium containing 300mg/L carbenicillin for delayed culture for 7 days, then transferring the callus to a differentiation culture medium containing 50mg/L kanamycin and 300mg/L carbenicillin for differentiating cluster buds and carrying out screening culture to obtain a positive explant, and finally transferring the positive explant to a rooting and seedling strengthening culture medium containing 50mg/L kanamycin and 300mg/L carbenicillin for culture. The overall regeneration rate reaches 36.7%, and the positive rate reaches 12.1%.

The preparation method of the agrobacterium infection solution comprises the following steps: selecting single agrobacterium colony, placing the single agrobacterium colony in a 1.5ml centrifuge tube filled with 1ml YEB liquid culture medium, placing the centrifuge tube in a shaking table at 28 ℃, culturing for 8h at 200 rpm, transferring all bacteria liquid to a 100ml triangular flask filled with 50ml YEB liquid culture medium containing 200mg/L AS under an aseptic environment, placing the triangular flask in the shaking table at 28 ℃, culturing at 200 rpm until the od value of the agrobacterium is =0.8, and sealing the triangular flask by using a breathable sealing film in the process of culturing the agrobacterium. The bacterial solution was centrifuged at 5000rpm for 5min in a sterilized centrifuge tube, the supernatant was decanted, resuspended to od =0.6 using 1/2MS liquid medium containing 3% sucrose +200mg/LAS, and then allowed to stand at room temperature for 3h.

The growth medium is MSM + 0.25mg/L TDZ + 0.1 mg/L IBA + 2wt.% sucrose; the callus induction culture medium MSM + 0.25mg/L TDZ +0.5 mg/L IBA + 2wt.% sucrose; the differentiation medium is MSM +0.5 mg/L TDZ + 0.1 mg/L IBA + 2wt.% sucrose; the rooting and seedling strengthening culture medium is MSM + 2wt.% of sucrose. Comparative experiments found that all explants died in the sucrose-free medium and the plant growth state improved with increasing sucrose concentration until the sucrose concentration was 2%. However, when the sucrose concentration was increased to 3%, growth was significantly inhibited (FIG. 1). And trace amount of growth regulator can increase the growth rate of the plant, but with the increase of the concentration of cytokinin and auxin, the growth rate of the explant is reduced. For example, as the concentration of cytokinin increases, the growth rate of the explant decreases and the internode distance significantly decreases. In addition, when the concentration of auxin IBA reaches 0.5mg/L, green compact massive callus is formed at the base of the segment, and the callus is more obvious with the increase of the concentration of IBA (FIG. 2).

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims

1. An isolated rapid propagation and genetic transformation method for passion fruit is characterized in that: the method comprises the following steps:

(1) Rapid bulk harvest of sterile explants: peeling seeds from fruits, cleaning, soaking the seeds for 72h, planting the seeds in a 128-hole tray one by one hole, and taking autoclaved pure vermiculite as a substrate; placing the plug disc in an environment at 28-32 ℃, and germinating 15 days later after the seeds are germinated; cutting off the embryonic axis to the stem tip part when the cotyledon is just unfolded, washing the cotyledon until the cotyledon is clean, soaking the cotyledon in saturated washing powder water for 20min, then washing the cotyledon with running water for 30min, and finishing the subsequent steps in a superclean workbench; soaking the material in 75% w/v alcohol for 30s, washing with sterile double distilled water for 2 times, soaking with 7.5% w/v sodium hypochlorite solution for 10min while shaking the bottle body, washing with sterile double distilled water for 3-6 times, and inoculating the explant to growth medium for growth; after 30 days, the bud seedlings grow up, and the internode stems of the aseptic seedlings are cut out to be used as fast propagation explants;

(2) The in vitro rapid propagation method comprises the following steps: inoculating the fast propagation explant into a callus induction culture medium for culturing for 7 days, then transferring the callus induction culture medium into a differentiation culture medium for inducing cluster buds, cutting off the cluster buds, transferring the cut cluster buds into a rooting and seedling strengthening culture medium for culturing to obtain a tissue culture seedling; opening a cap of a tissue culture seedling growing strongly 2 days before transplanting for hardening the seedling, washing a root residual culture medium with tap water before transplanting, and then transplanting the seedling into a mixed matrix with the mass ratio of peat soil to perlite being 7: 3 for culturing to obtain an aseptic seedling;

(3) The genetic transformation method comprises the following steps: taking the stem section of the aseptic seedling in the step (2) as an infection material, pre-culturing the infection material in a callus induction culture medium for 3 days, soaking the infection material in an agrobacterium infection solution for 15min during infection, and continuously shaking the bottle body during infection; then transferring the callus to a callus induction culture medium without any antibiotic for co-culture for 3 days, transferring the callus to a callus induction culture medium containing 300mg/L carbenicillin for delayed culture for 7 days, transferring the callus to a differentiation culture medium containing 50mg/L kanamycin and 300mg/L carbenicillin for differentiating cluster buds and carrying out screening culture to obtain a positive explant, and finally transferring the positive explant to a rooting and seedling strengthening culture medium containing 50mg/L kanamycin and 300mg/L carbenicillin for culture;

the growth medium is MSM + 0.25mg/L TDZ + 0.1 mg/L IBA + 2wt.% sucrose; the callus induction culture medium is MSM + 0.25mg/L TDZ +0.5 mg/L IBA + 2wt.% cane sugar; the differentiation medium is MSM +0.5 mg/L TDZ + 0.1 mg/L IBA + 2wt.% sucrose; the rooting and seedling strengthening culture medium is MSM + 2wt.% of sucrose.