CN116897834A - A kind of regeneration method and genetic transformation method of Laohan melon - Google Patents

Abstract

The present invention is a regeneration method and genetic transformation method of old Han melon. A method for regeneration of old Han melon, including the following steps: (1) disinfection of seeds and acquisition of sterile explants; (2) induction and culture of callus; (3) induction and culture of adventitious buds; (4) adventitious buds Elongation of buds; (5) Induction culture of roots. The invention also discloses a genetic transformation method of Laohan melon. The regeneration method and genetic transformation method of Laohan melon described in the present invention successfully realize the establishment of high-efficiency in vitro regeneration and genetic transformation system of Laohan melon cotyledons, so that the genetic transformation rate is above 86%.

Description

Regeneration method and genetic transformation method of cantaloupe

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a regeneration method and a genetic transformation method of cantaloupe.

Background

Melon (cucumeleo l.) belongs to cucurbitaceae cucumber genus, is a deeply favored fruit, and "the old melon (academic name of schek melon)" is a special product in Xinjiang, has the characteristics of softness, delicious taste, high water content, high sugar content and the like, is very suitable for old people and children to eat, and is deeply popular with wide consumers. "Laohan melon" has the characteristics of heat-sensitive raw materials, and is mainly sold in fresh. Because the softening speed is high in the storage and transportation processes, the problems of storage and transportation are caused, and the economic benefits of farmers are greatly lost.

As a special product in Xinjiang, the cantaloupe is extremely inadvisable to store due to the sweet, soft and delicious property. Means of genetic engineering are urgently needed to improve the quality thereof. Through genetic transformation technology, the genome of melon is changed, the purpose of directional improvement is achieved, and a new way is provided for breeding of the cantaloupe.

At present, with the progress of the related art, it has become realistic to cultivate new varieties with excellent traits by genetic transformation methods. With the continuous deep research of transgenosis, the character which can be changed is not limited to stress resistance, insect resistance or disease resistance, but also extends to the aspects of quality improvement and the like. Although genetic transformation of melon has been studied at present, the genetic transformation of different genotypes is greatly different. Based on the above, no suitable genetic transformation system can be used for directionally transforming the cantaloupe.

Disclosure of Invention

In order to solve the technical problems, the invention provides a regeneration method of the gerhan melon, which successfully realizes the efficient in-vitro regeneration of the leaf of the gerhan melon.

In order to achieve the above purpose, the technical scheme adopted is as follows:

a regeneration method of a cantaloupe comprises the following steps:

(1) Seed sterilization and sterile explant acquisition: removing shells of the seeds of the Laohuan melon, sterilizing, culturing in a germination culture medium until the leaves of the Laohuan melon are completely unfolded and the true leaves are not grown, thus obtaining a sterile explant;

(2) Induction culture of callus: cutting off the head and tail of the sterile explant, inoculating a callus induction culture medium, and culturing for 15-20 days under the culture condition of 25-28 ℃;

(3) Induction culture of adventitious buds: after the callus appears, transferring the explant into a bud induction culture medium, and culturing until adventitious buds appear;

(4) Elongation of adventitious bud: when the length of the adventitious bud is 0.5-1cm, inoculating the explant with the adventitious bud into an adventitious bud elongation culture medium for culture;

(5) Induction culture of roots: when the adventitious buds are elongated to 1.5-3cm, cutting off the adventitious buds, and inoculating the adventitious buds into a root induction culture medium for culture.

Further, in the step (1), the germination medium is: adding 15-20g/L sucrose and 8-9g/L agar strips into 1/2MS culture medium, and adjusting pH to 5.8-6.0;

in the steps (2) - (3), the culture medium is as follows: adding 30g/L sucrose, 9g/L agar, 0.5-1.0 mg/L6-BA, 0.15-0.3mg/L IAA into MS culture medium, and pH value is 5.8-6.0;

in the step (4), the adventitious bud elongation culture medium is: adding 30g/L sucrose, 9g/L agar, 0.5-1.0 mg/L6-BA, 0.15-0.3mg/L IAA, 0.5-1.0mg/LGA3 into MS culture medium, and pH value is 5.8-6.0;

in the step (5), the root induction culture medium is: 15g/L sucrose, 9g/L agar strips and 0.6-0.9mg/L IAA are added into the 1/2MS culture medium, and the pH value is 5.8-6.0.

Still further, in the step (1), the germination medium is: 15g/L sucrose and 8g/L agar strips are added into a 1/2MS culture medium, and the pH value is 5.8;

in the steps (2) - (3), the culture medium is as follows: 30g/L sucrose, 9g/L agar strips, 1.0 mg/L6-BA and 0.2mg/L IAA are added into an MS culture medium;

in the step (4), the adventitious bud elongation culture medium is: 30g/L sucrose, 9g/L agar, 1.0 mg/L6-BA, 0.3mg/L IAA and 1.0mg/LGA3 are added into the MS culture medium;

in the step (5), the root induction culture medium is: 1/2MS culture medium, 15g/L sucrose, 9g/L agar strips, 0.8mg/L IAA.

The invention also aims to provide a genetic transformation method of the cantaloupe, which can successfully obtain the genetically transformed cantaloupe seedlings, and the genetic transformation efficiency reaches 86%.

In order to achieve the above purpose, the technical scheme adopted is as follows:

a genetic transformation method of cantaloupe, comprising the following steps: after the regeneration method is adopted, the identification of the genetic transformation seedlings is carried out;

wherein, the step (2) is to perform preculture, infection and co-culture of explants before the callus induction culture, and then perform the callus induction culture;

the pre-culture process of the explant comprises the following steps: cutting off the head and tail of the sterile explant, inoculating the sterile explant into a preculture medium, and performing dark culture under the culture condition of 25-28 ℃;

the infection and co-culture process comprises the following steps: infecting the precultured Laohuan melon cotyledon with agrobacterium tumefaciens bacteria liquid carrying plant expression vector, transferring to callus inducing culture medium, separating the explant from the culture medium with sterile filter paper, and co-culturing;

in the genetic transformation method, in the induction culture of the adventitious buds in the step (3): the adventitious bud induction culture also comprises timentin and an antibiotic hygromycin B;

elongation of adventitious buds in step (4) in the genetic transformation method: the adventitious bud elongation culture medium also comprises timentin and antibiotics hygromycin B;

the genetic transformation method comprises the following steps of (5) root induction culture: the root induction culture medium also comprises timentin and the antibiotic hygromycin B;

identification of the genetically transformed seedlings: GFP fluorescence detection is carried out on the genetically transformed Laohuan seedlings after the induction culture of the roots; extracting genome DNA of genetically transformed Laohanganese melon seedling, PCR positive detection and screening positive genetically transformed seedling.

In the step (2), the agrobacterium tumefaciens bacteria solution is formed by resuspending agrobacterium tumefaciens carrying a plant expression vector in an MS liquid culture medium, wherein the MS liquid culture medium comprises the following components: MS basic powder, 15g/L sucrose, 15g/L glucose, pH value is 5.8;

the culture medium for the preculture is MS culture medium, 30g/L sucrose, 9g/L agar, 0.25mg/L6-BA and 0.15mg/L IAA;

in the step (3), the concentration of the timentin is 200-250mg/L, and the concentration of the hygromycin is 10-20mg/L;

in the step (4), the concentration of the timentin is 200-250mg/L, and the concentration of the hygromycin is 10-20mg/L;

in the step (4), the concentration of the timentin is 200-250mg/L, and the concentration of the hygromycin is 10-20mg/L.

Still further, in the step (2), the agrobacterium tumefaciens bacteria solution also contains 50-100mg/L acetosyringone.

Still further, the OD of the agrobacterium tumefaciens bacterial solution ₆₀₀ 0.4-0.6.

Furthermore, the agrobacterium tumefaciens is agrobacterium tumefaciens GV3101, and the plant expression vector is pCAMBIA1302.

Further, in the step (2), the infection and co-culture process is performed for 2 days under the culture condition of 25-28 ℃;

in the step (3), culturing is carried out in a tissue culture chamber with the photoperiod of 16h illumination/8 h darkness and the illumination intensity of 5000lx at the temperature of 25-28 ℃ and the culture medium is replaced for 15-20 days;

in the step (4), culturing is carried out in a tissue culture chamber with the photoperiod of 16h illumination/8 h darkness and the illumination intensity of 5000lx at the temperature of 25-28 ℃ and the culture medium is replaced for 15-20 days;

in the step (5), culturing is carried out in a tissue culture room with the photoperiod of 16h illumination/8 h darkness and the illumination intensity of 5000lx at the temperature of 25-28 ℃.

Compared with the prior art, the invention has the following advantages:

according to the method for in-vitro regeneration and genetic transformation of the Laohan melon cotyledon, a group of hormone ratios with very high regeneration frequency is obtained through screening. On the basis of establishing a high-efficiency regeneration system, a high-efficiency genetic transformation system of the cantaloupe is established, and the genetic transformation rate is kept at about 86%. The method has higher practicability and high efficiency on subsequent genetic engineering breeding of the cantaloupe.

Drawings

FIG. 1 acquisition of sterile explants of Laohuan;

FIG. 2 induced culture of Calophyllum Inophyllum callus;

FIG. 3 is a graph showing the formation of resistant adventitious buds of Laohan melon;

FIG. 4 is an elongation chart of adventitious buds of Laohan melon;

FIG. 5 is a rooting chart of the cantaloupe;

FIG. 6 shows a GFP fluorescence detection diagram of the cantaloupe;

FIG. 7 shows PCR molecular detection of Laohanggua.

Detailed Description

In order to further illustrate the regeneration method and genetic transformation method of the cantaloupe according to the present invention, the following describes the specific implementation, structure, characteristics and efficacy of the regeneration method and genetic transformation method of the cantaloupe according to the present invention in detail with reference to the preferred embodiments. In the following description, different "an embodiment" or "an embodiment" do not necessarily refer to the same embodiment. Furthermore, the particular features, structures, or characteristics of one or more embodiments may be combined in any suitable manner.

The regeneration method and genetic transformation method of the invention will be described in further detail with reference to specific examples:

the invention belongs to the technical fields of genetic engineering and plant tissue culture, and relates to a method for establishing an efficient regeneration and genetic transformation system of a gerhan melon seed leaf. The old melon is a typical respiratory-transition type fruit, is a melon variety which is extremely not stored, and is extremely easy to be overripe, softened and spoiled in the processes of transportation, preservation, sales and the like after picking. The invention aims at lacking research on genetic transformation systems of the cantaloupe. Provides a novel method for high-efficiency regeneration of the Laohan melon seed leaf and establishment of a genetic transformation system, the method successfully obtains the Laohan melon seed seedling subjected to genetic transformation, and the genetic transformation efficiency reaches 86 percent.

Agrobacterium-mediated methods are currently the primary method of dicotyledonous plant inheritance and partial monocotyledonous plant transformation. Fang and Grumet reported for the first time the use of agrobacterium tumefaciens mediated pouring of neomycin transferase N-PT ii into melon. Compared with other methods, the agrobacterium-mediated method is mature, simple to operate and low in cost. The specific technical scheme is as follows:

a regeneration method of a cantaloupe comprises the following steps:

(1) Seed sterilization and sterile explant acquisition: removing shells of the seeds of the Laohuan melon, sterilizing, culturing in a germination culture medium until the leaves of the Laohuan melon are completely unfolded and the true leaves are not grown, thus obtaining a sterile explant;

(2) Induction culture of callus: cutting off the head and tail of the sterile explant, inoculating a callus induction culture medium, and culturing for 15-20 days under the culture condition of 25-28 ℃;

(3) Induction culture of adventitious buds: after the callus appears, transferring the explant into a bud induction culture medium, and culturing until adventitious buds appear;

(4) Elongation of adventitious bud: when the length of the adventitious bud is 0.5-1cm, inoculating the explant with the adventitious bud into an adventitious bud elongation culture medium for culture;

(5) Induction culture of roots: when the adventitious buds are elongated to 1.5-3cm, cutting off the adventitious buds, and inoculating the adventitious buds into a root induction culture medium for culture.

Preferably, in the step (1), the germination medium is: adding 15-20g/L sucrose and 8-9g/L agar strips into 1/2MS culture medium, and adjusting pH to 5.8-6.0;

in the steps (2) - (3), the culture medium is as follows: adding 30g/L sucrose, 9g/L agar, 0.5-1.0 mg/L6-BA, 0.15-0.3mg/L IAA into MS culture medium, and pH value is 5.8-6.0;

in the step (4), the adventitious bud elongation culture medium is: adding 30g/L sucrose, 9g/L agar, 0.5-1.0 mg/L6-BA, 0.15-0.3mg/L IAA, 0.5-1.0mg/LGA3 into MS culture medium, and pH value is 5.8-6.0;

in the step (5), the root induction culture medium is: 15g/L sucrose, 9g/L agar strips and 0.6-0.9mg/L IAA are added into the 1/2MS culture medium, and the pH value is 5.8-6.0.

Further preferably, in the step (1), the germination medium is: 15g/L sucrose and 8g/L agar strips are added into a 1/2MS culture medium, and the pH value is 5.8;

in the steps (2) - (3), the culture medium is as follows: 30g/L sucrose, 9g/L agar strips, 1.0 mg/L6-BA and 0.2mg/L IAA are added into an MS culture medium;

in the step (4), the adventitious bud elongation culture medium is: 30g/L sucrose, 9g/L agar, 1.0 mg/L6-BA, 0.3mg/L IAA and 1.0mg/LGA3 are added into the MS culture medium;

in the step (5), the root induction culture medium is: 1/2MS culture medium, 15g/L sucrose, 9g/L agar strips, 0.8mg/L IAA.

A genetic transformation method of cantaloupe, comprising: after the regeneration method is adopted, the identification of the genetic transformation seedlings is carried out; specific:

(1) Seed sterilization and sterile explant acquisition in the regeneration process described above;

(2) Preculture of explants: cutting off the head and tail of the sterile explant, inoculating the sterile explant into a preculture medium, and performing dark culture under the culture condition of 25-28 ℃;

infection and co-cultivation: the agrobacteria liquid of the root cancer carrying the plant expression vector is used for invading the dark-cultured Laohuan melon cotyledon in the step (2), then the dark-cultured Laohuan melon cotyledon is transferred to an original culture medium, and an aseptic filter paper is used for separating the explant from the culture medium, and the culture condition is the same as the culture time in the step (2);

(3) The callus induction culture in the regeneration method is characterized in that the culture condition is changed into dark culture, and the culture time is shortened to 2 days;

(4) Induction culture of adventitious buds in the regeneration method described above, but is different in that the antibacterial component of timentin and the antibiotic hygromycin B are added to the adventitious bud induction culture of claim 1;

(5) The method for regenerating the adventitious bud comprises the step of elongating the adventitious bud, wherein the antibacterial ingredient, such as timentin and hygromycin B, is added to the adventitious bud of claim 1;

(6) Induction culture of roots in the regeneration method as described above, but is different in that the antibacterial component, timentin and the antibiotic hygromycin B, are added to the root induction culture of claim 1;

(7) Identification of genetically transformed seedlings: performing GFP fluorescence detection on the genetically transformed Laohanggua seedlings in the step (6); extracting genome DNA of genetically transformed Laohanganese melon seedling, PCR positive detection and screening positive genetically transformed seedling.

Preferably, in the step (2), the agrobacterium tumefaciens bacterial solution is formed by resuspending agrobacterium tumefaciens carrying a plant expression vector in an MS liquid culture medium, and the MS liquid culture medium comprises the following components: MS basic powder, 15g/L sucrose, 15g/L glucose, pH value is 5.8;

the culture medium for the preculture is MS culture medium, 30g/L sucrose, 9g/L agar, 0.25mg/L6-BA and 0.15mg/L IAA;

in the step (3), the concentration of the timentin is 200-250mg/L, and the concentration of the hygromycin is 10-20mg/L;

in the step (4), the concentration of the timentin is 200-250mg/L, and the concentration of the hygromycin is 10-20mg/L.

In the step (2), the agrobacterium tumefaciens bacteria solution also contains 50-100mg/L acetosyringone.

Further preferably, the OD of the agrobacterium tumefaciens bacterial solution ₆₀₀ 0.4-0.6.

Preferably, the agrobacterium tumefaciens is agrobacterium tumefaciens GV3101, and the plant expression vector is pCAMBIA1302.

Preferably, in the step (2), the infection and co-culture process is performed for 2 days under the culture condition of 25-28 ℃;

in the step (3), culturing is carried out in a tissue culture chamber with the photoperiod of 16h illumination/8 h darkness and the illumination intensity of 5000lx at the temperature of 25-28 ℃ and the culture medium is replaced for 15-20 days;

in the step (4), culturing is carried out in a tissue culture chamber with the photoperiod of 16h illumination/8 h darkness and the illumination intensity of 5000lx at the temperature of 25-28 ℃ and the culture medium is replaced for 15-20 days;

in the step (5), culturing is carried out in a tissue culture room with the photoperiod of 16h illumination/8 h darkness and the illumination intensity of 5000lx at the temperature of 25-28 ℃.

Compared with the prior art related to the same melon, the method is more suitable for the variety of the cantaloupe in terms of the variety, content and proportion of the plant growth regulator in the culture medium.

The technical scheme of the invention is conventional in the art unless specifically stated otherwise.

The plant expression vector used in the invention is PCAMBIA1302, and the agrobacterium tumefaciens is Agrobacterium tumefaciens GV3101.

MS medium refers to commercially available MS base powder, and the dosage is 4.4g/L.1/2MS medium refers to half-strength MS medium, and the dosage is 2.2g/L.

Example 1: efficient in-vitro regeneration method for folium momordicae

The specific operation steps are as follows:

(1) Seed disinfection and sterile explant acquisition

Selecting full and disease-spot-free Laohuan seeds, peeling off seed shells, peeling a certain number of seeds, placing the seeds into a clean conical flask for standby, pouring the seeds for standby into a sterilized conical flask in a sterile operation table, firstly cleaning the seeds once with sterile water, and washing away small impurities. Then, the mixture was sterilized with 75% alcohol for 30 seconds (the time for sterilization should be strict), and after washing with sterilized water twice, the mixture was sterilized with 2% sodium hypochlorite for 10 minutes. Finally, cleaning the mixture for 3 to 4 times by using sterile water.

The sterilized seeds are placed on sterile filter paper, the surface of the sterilized seeds are dried in the air, and then the sterilized seeds are sown in a germination culture medium, wherein the sterilized seeds are as follows: 15g/L sucrose and 8g/L agar strips were added to half strength MS medium (1/2 MS medium) at pH 5.8. The seeds are cultivated in a tissue culture room with the temperature of 25-28 ℃ and the illumination intensity of 5000lx-8000lx and the photoperiod of 16h illumination/8 h after being dark cultivated for two days. When melon seeds grow until cotyledons develop and true leaves do not grow, the cotyledons of the cantaloupe in the state are taken as explants (figure 1).

(2) Influence of 6-BA and IAA concentrations on callus induction and adventitious buds

Cotyledons of 4-6d were selected as explants, the top and base of the cotyledons were excised, and then cut into two parts from the middle. The leaves were inoculated right side up into medium (MS medium with 30g/L sucrose, 9g/L agar strips, and different concentrations of 6-BA and IAA, pH 6.0) containing different concentrations of 6-BA and IAA for cultivation, 12 different hormone ratios were designed in total. There were 10 plates per hormone mix, with 6 explants per plate. Culturing in a tissue culture room with temperature of 26deg.C, photoperiod of 16h light/8 h darkness and light intensity of 8000lx, observing the explant callus rate about 25d, and counting adventitious bud differentiation rate.

Table 1 6-influence of BA and IAA on the differentiation of adventitious buds of Laohan melon cotyledons

Different concentrations of 6-BA and IAA affect their induction of adventitious buds. The results are shown in Table 1, and the concentration of the medium was 1.0mg.L ^-1 6-BA and 0.2 mg.L of (C) ^-1 IAA has the highest induction rate of callus and adventitious bud, and the induction rate is 91.66%. In the explant, the induction rate of the callus is 100% in other hormone ratios, but the differentiation rate of the adventitious buds is different, and the growth state of the adventitious buds is also different. Thus, the culture medium used was: the MS culture medium is added with 30g/L sucrose, 9g/L agar, 1.0 mg/L6-BA and 0.2mg/L IAA as the optimal callus and bud induction culture medium.

(3) Elongation of adventitious bud

When the induced buds grow to about 0.5-1.0cm, cutting off and inoculating on an elongation culture medium, wherein the elongation culture medium is as follows: MS medium of hormone 1.0 mg/L6-BA, 0.3mg/L IAA and 1mg/L GA3, 30g/L sucrose and 9g/L agar strips, pH 6.0.

(4) Determination of adventitious bud rooting culture medium

After the adventitious bud was elongated for about 14d, when the adventitious bud was about 2-3cm in length (FIG. 3), it was cut out from the basal part and inoculated into an induction medium (root induction medium: half-strength MS medium added with 15g/L sucrose, 9g/L agar bars, 0.2-0.9mg/L IAA, pH 6.0) to which roots of different types and concentrations (0.1, 0.2 and 0.5 mg/L) were added. Counting the days, rooting rate and rooting number of the roots, and screening out the optimal rooting culture medium formula. The experiment was repeated three times. And selecting the most suitable rooting condition of the cantaloupe to carry out a subsequent genetic transformation experiment. The results are shown in Table 2.

TABLE 2 Effect of IAA (indoleacetic acid) at different concentrations on rooting of adventitious buds of Haemonchus arvensis

As can be seen from Table 2, when IAA with different concentrations is added into the culture medium, adventitious buds can root, but the concentrations are different, and when IAA concentration is 0.6-0.9mg/L, the rooting rate is more than 60%. Wherein, when 0.8mg/L IAA is added into the culture medium, the rooting effect is best, and the rooting rate reaches 80%. Therefore, the best rooting medium is 1/2MS medium added with 15g/L sucrose, 9g/L agar strips and 0.8mg/L IAA.

Example 2.

The specific operation steps are as follows:

(1) Seed sterilization and sterile explant acquisition:

the sterilization process of the seeds was the same as in step (1) of example 1.

The sterilized seeds are placed on sterile filter paper, the surface of the sterilized seeds are dried in the air, and then the sterilized seeds are sown in a germination culture medium, wherein the sterilized seeds are as follows: a half-strength MS medium (1/2 MS medium) was added with 20g/L sucrose and 9g/L agar bars at pH 6.0. The seeds were cultivated in a tissue culture room at 26℃and an illumination intensity of 5000lx-7000lx with a photoperiod of 16h illumination/8 h after dark cultivation for two days. When the melon seeds grow until the cotyledons are unfolded and the true leaves are not grown, taking the cotyledons of the cantaloupe in the state as explants.

(2) Induction culture of callus and adventitious bud

The head and tail of the aseptic explant are cut off, and the aseptic explant is inoculated with a callus induction medium and cultured for 20 days under the culture condition of 25 ℃.

After the callus appears, the explant is transferred into a bud induction culture medium and cultured until adventitious buds appear.

The culture conditions for the induction culture of the callus and the adventitious bud are as follows: culturing in a tissue culture room with 25 deg.C and photoperiod of 16 h/8 h darkness and illumination intensity of 8000lx, and observing the cure rate of explant about 25 d.

The induction culture of the callus and the adventitious bud adopts a culture medium as follows: to the MS medium were added 30g/L sucrose, 9g/L agar, 1.0 mg/L6-BA, 0.2mg/L IAA.

(3) Elongation of adventitious bud

When the induced buds grow to about 0.5-1.0cm, cutting off and inoculating on an elongation culture medium, wherein the elongation culture medium is as follows: MS medium of hormone 0.5 mg/L6-BA, 0.15mg/L IAA and 0.5mg/L GA3, 30g/L sucrose and 9g/L agar strips.

(4) Determination of adventitious bud rooting culture medium

After the adventitious bud is elongated for about 14d, when the length of the adventitious bud is about 1.5-3cm, the adventitious bud is cut out from the basal part, and the inoculated root induction medium (the root induction medium is half-strength MS medium, 15g/L sucrose, 9g/L agar, 0.8mg/L IAA and pH value is 5.8) is added for culture.

Example 3.

The specific operation steps are as follows:

(1) Seed sterilization and sterile explant acquisition:

the sterilization process of the seeds was the same as in step (1) of example 1.

The sterilized seeds are placed on sterile filter paper, the surface of the sterilized seeds are dried in the air, and then the sterilized seeds are sown in a germination culture medium, wherein the sterilized seeds are as follows: 18g/L sucrose and 8.5g/L agar strips were added to half strength MS medium (1/2 MS medium) at pH 5.9. The seeds are cultivated in a tissue culture room with the temperature of 25 ℃ and the illumination intensity of 6000lx-8000lx and the light period of 16h illumination/8 h after being dark cultivated for two days. When the melon seeds grow until the cotyledons are unfolded and the true leaves are not grown, taking the cotyledons of the cantaloupe in the state as explants.

(2) Induction culture of callus and adventitious bud

The head and tail of the aseptic explant are cut off, and the aseptic explant is inoculated with a callus induction medium and cultured for 18 days under the culture condition of 28 ℃.

After the callus appears, the explant is transferred into a bud induction culture medium and cultured until adventitious buds appear.

The culture conditions for the induction culture of the callus and the adventitious bud are as follows: culturing in a tissue culture room with 28 deg.C and 16 h/8 h dark photoperiod and 8000lx illumination, and observing the cure rate of explant about 25 d.

The induction culture of the callus and the adventitious bud adopts a culture medium as follows: 30g/L sucrose, 9g/L agar, 0.8 mg/L6-BA, 0.2mg/L IAA and pH 5.9 were added to the MS medium.

(3) Elongation of adventitious bud

When the induced buds grow to about 0.5-1.0cm, cutting off and inoculating on an elongation culture medium, wherein the elongation culture medium is as follows: MS medium of hormone 0.7 mg/L6-BA, 0.20mg/L IAA and 0.6mg/L GA3, 30g/L sucrose and 9g/L agar strips, pH 5.9.

(4) Determination of adventitious bud rooting culture medium

After the adventitious bud was elongated for about 14d, the adventitious bud was cut out from the basal part until the length of the adventitious bud reached about 2 to 3cm, and the culture was performed in an induction medium (the induction medium for root is a half-strength MS medium, 15g/L sucrose, 9g/L agar, 0.8mg/L IAA, and pH of 5.9) added thereto.

Example 4: agrobacterium-mediated high-efficiency genetic transformation method for folium nelumbinis

The specific operation steps are as follows:

time of Pre-culture of explants

The proper preculture enables the explant (obtained by adopting the step (1) of the embodiment 1) which is just separated from the plant to better regulate the physiological state so as to quicken the cell division, and the cells need a certain time to finish the generation and release of signal molecules such as phenolic compounds and the like in the process of wound healing of the plant, so that the agrobacterium infection is better induced into the explant, and therefore, the proper preculture can improve the genetic transformation efficiency of the plant.

The Laohuan cotyledon (with head and basal part removed) is used as a genetic transformation receptor, inoculated on a preculture medium, and the composition is MS culture medium, sucrose, agar, 0.25mg/L6-BA and IAA, and the culture medium is dark-cultured at 25-28 ℃ for 2 days.

Preparation of B expression vector electric shock method for introducing agrobacterium and agrobacterium tumefaciens bacterial liquid

The pCAMBIA1302 vector was first transferred into the competent Agrobacterium GV3101 by electroporation as follows:

the electric shock cup is firstly soaked in 70% alcohol, is firstly washed three times by sterile water, is taken out, is rinsed multiple times by sterile water, is put on the electric shock cup by filter paper prepared in advance, is reversely put for blow drying, and is collected by 2ml of bacterial liquid (5000 r,2 min) when the bacterial grows to the logarithmic growth phase, namely the bacterial activity is maximum (OD value is about 0.6, bacterial liquid starts to be changed from wine red to orange).

Adding 200u of sterile water, re-suspending, adding 5ul of plasmid, mixing, and ice-bathing for 30min; filling the electric shock cup into disposable gloves which are trimmed in advance, and precooling for 30min; adding 1ml of bacterial liquid into the electric shock cup, suspending the bacterial liquid drop by drop, adding the bacterial liquid in the middle, putting the electric shock cup onto an electrotransport device, and electrically shocking for 1S; after the electric shock, l ml of LB diluted bacteria liquid is added into an electric shock cup, and the bacterial liquid is transferred to a new centrifuge tube.

Cleaning the electric shock cup.

200ul of bacterial liquid is coated on a plate, and the plate is reversely cultured at 28 ℃ for 42 hours in a dark place. When single colonies were grown, the single colonies were picked for PCR detection, positive colonies were determined, and Agrobacterium identified as positive bacterial liquid was inoculated into LB liquid medium containing 50mg/L kanamycin, 50mg/L gentamicin and 100mg/L rifampicin, and cultured overnight at 28℃at 150 rpm. Sterilizing with 40% glycerol, and storing in refrigerator at-80deg.C.

C infection and co-cultivation

Infection: the previous night the Agrobacterium solution containing the PCAMBIA1302 vector was inoculated in 1% inoculum size into LB liquid medium containing 50mg/L kanamycin, 50mg/L gentamicin and 100mg/L rifampicin.

When the concentration of the bacterial liquid was 0.5, the bacterial liquid was poured into a sterile 50ml centrifuge tube, and the bacterial liquid was collected by centrifugation at 5000rpm for 10min, and the bacterial liquid was resuspended in MS liquid medium (containing 15g/L sucrose, 15g/L white granulated sugar, pH 5.8), and 50mg/L Acetosyringone (AS) was added to the resuspension.

Sealing the bottle mouth of the conical flask containing the heavy suspension, and placing the conical flask into a shaking table at 180rpm at 28 ℃ for induction for 30-60min. At the same time, melon explants after two days of dark culture were clamped into a sterile empty conical flask using sterile forceps.

Co-cultivation: after all explants were transferred to Erlenmeyer flasks, a layer of sterile filter paper was laid on the pre-incubated plates. Pouring the strain into a sterile bottle containing the explant after the strain is induced, pouring out the heavy suspension after 10min of infection, clamping the explant on filter paper by using sterile forceps, and sucking out more strain around the explant.

Naturally airing for 5min, and then spreading in a preculture medium containing a layer of filter paper. Followed by co-cultivation in the dark at 25-28℃for two days.

The medium used for the dark culture was MS medium supplemented with 30g/L sucrose, 9g/L agar bar, 0.50 mg/L6-BA and 0.15mg/L IAA, pH 5.8.

Screening culture of resistant callus and resistant buds

After co-culture, transferring the explant into a callus and bud induction culture medium, wherein the callus and bud induction culture medium comprises MS culture medium added with 30g/L sucrose, 9g/L agar strip, 0.5-1.0 mg/L6-BA and 0.15-0.3mg/L IAA, 200mg/L timentin and 10mg/L hygromycin, and the pH value is 5.8.

The culture conditions were 26℃and the photoperiod was 16h light/8 h dark, the light intensity was 5000lx, and 20d was changed to a single medium, which was capable of forming both callus (FIG. 2) and adventitious buds.

Elongation culture of E-resistant adventitious buds

When the adventitious bud length reached around 1cm (FIG. 3), the explant was transferred to a bud elongation medium. The bud elongation culture medium is MS culture medium, 30g/L sucrose+9 g/L agar bar+0.5 mg/L6-BA and 0.15mg/L IAA+1mg/LGA3 are added, 200mg/L timentin+10 mg/L hygromycin is also added, and the pH value is 5.8.

The culture condition is 26 ℃, the photoperiod is 16h illumination/8 h darkness, and the illumination intensity is 5000lx.

F rooting of resistant adventitious buds

When the adventitious bud is 2-3cm long (FIG. 4), the adventitious bud is cut off by using a sterile scissors or by using a sterile planing tool. The excised adventitious buds are placed in rooting medium. The rooting culture medium is 1/2MS culture medium, 15g/L sucrose+9 g/L agar strips+0.6-0.9 mg/L IAA, 200mg/L timentin+10 mg/L hygromycin and the pH value is 5.8.

Culture conditions: culturing is carried out in a tissue culture room with the light period of 16h illumination/8 h darkness and the illumination intensity of 5000lx at 26 ℃.

PCR molecular detection of G genetic transformation seedlings

When the adventitious bud length is 6-8cm and the root system is 4-7 roots (figure 5), the aseptic seedling is subjected to seedling training and transplanting.

After the aseptic seedlings are trained to survive, DNA of a genetic transformation strain is extracted by using a DNAsecure novel plant genome DNA extraction kit (Tiangen), and PCR amplification is carried out on 700bp GFP fragments by using primers GFP-F5'-GGTCTAGAATGGTGAGCAGGGCG-3' and GFP-R5'-GGCTGCAGTTACTTGTACAGCTCGTCCATG-3' by taking the genomic DNA of the Lao Han Guo as a template.

The PCR reaction procedure was: 94 ℃ for 5min;94 ℃ for 30s; 30s at 55 ℃; 45s at 72℃C (35 cycles in this section); 7min at 72 ℃. PCR was performed using untransformed genomic DNA from Laohuan melon as a template as a negative control. 10ul of the PCR product was used for agarose gel electrophoresis detection. The results are shown in FIG. 7. The GFP fluorescence detection chart of the Laohan melon is shown in FIG. 6.

Example 5.

The specific operation steps are as follows:

pre-culture of explants: as in step A of example 4, the culture was performed at 26℃for 2 days in dark.

B, preparing agrobacterium tumefaciens bacterial liquid by introducing agrobacterium tumefaciens into an expression vector by an electric shock method: the same as in step B of example 4.

C infection and co-cultivation

The infestation procedure is identical to step C of example 4. The difference is that: 60mg/L Acetosyringone (AS) was added to the resuspension. Sealing the bottle mouth of the conical flask containing the heavy suspension, and placing the conical flask into a shaking table at 180rpm at 28 ℃ for 60min for induction.

Co-cultivation: after all explants were transferred to Erlenmeyer flasks, a layer of sterile filter paper was laid on the pre-incubated plates. Pouring the strain into a sterile bottle containing the explant after the strain is induced, pouring out the heavy suspension after 10min of infection, clamping the explant on filter paper by using sterile forceps, and sucking out more strain around the explant.

Naturally airing for 5min, and then spreading in a preculture medium containing a layer of filter paper. Followed by co-cultivation in the dark at 26℃for two days.

The medium used for the dark culture was MS medium supplemented with 30g/L sucrose, 9g/L agar bars, 1.0 mg/L6-BA and 0.30mg/L IAA, pH 6.0.

Screening culture of resistant callus and resistant buds

After the co-culture, the explants were transferred to callus and shoot induction medium for culture. The composition of the callus and bud induction culture medium is MS culture medium, 30g/L sucrose+9 g/L agar bar+0.6 mg/L6-BA and 0.20mg/L IAA are added, 250mg/L timentin+20 mg/L hygromycin is also added, and the pH value is 6.0.

The culture condition is 25 ℃, the photoperiod is 16h light/8 h dark, the light intensity is 5000lx, and the culture medium is replaced once for 15d, and can form not only callus but also adventitious buds.

Elongation culture of E-resistant adventitious buds

When the adventitious bud length reaches about 1cm, the explant is transferred to a bud elongation culture medium for culture. The bud elongation culture medium is MS culture medium, 30g/L sucrose+9 g/L agar bar+1 mg/L6-BA and 0.30mg/L IAA+0.8mg/LGA3 are added, 250mg/L timentin+20 mg/L hygromycin is also added, and the pH value is 6.0.

The culture conditions were 25℃and the photoperiod was 16h light/8 h dark, the light intensity was 5000lx,15d and the medium was changed once.

F rooting of resistant adventitious buds

When the length of the adventitious bud is 2-3cm, the adventitious bud is reduced by using a sterile scissors or cut by using a sterile planing knife. And placing the cut adventitious buds into a rooting medium for culturing. The rooting culture medium is 1/2MS culture medium, 15g/L sucrose, 9g/L agar strips, 0.8mg/L IAA, 250mg/L timentin, 20mg/L hygromycin and the pH value is 6.0.

Culture conditions: culturing is carried out in a tissue culture room with the light period of 16h illumination/8 h darkness and the illumination intensity of 5000lx at 25 ℃.

PCR molecular detection of G genetic transformation seedlings

When the adventitious bud length is 6-8cm and the root system is 4-7, the aseptic seedling is subjected to seedling hardening and transplanting.

After the aseptic seedlings are trained to survive, DNA of a genetic transformation strain is extracted by using a DNAsecure novel plant genome DNA extraction kit (Tiangen), PCR amplification is carried out on 700bp GFP fragments by using the genomic DNA of the Laohanganese as a template, and positive genetic transformation seedlings are screened.

Example 6.

The specific operation steps are as follows:

pre-culture of explants: as in step A of example 4, the culture was performed at 28℃for 2 days in dark culture.

B, preparing agrobacterium tumefaciens bacterial liquid by introducing agrobacterium tumefaciens into an expression vector by an electric shock method: the same as in step B of example 4.

C infection and co-cultivation

The infestation procedure is identical to step C of example 4. The difference is that: to the resuspension was added 50mg/L Acetosyringone (AS). Sealing the bottle mouth of the conical flask containing the heavy suspension, and placing the conical flask into a shaking table at 180rpm at 28 ℃ for 30min for induction.

Co-cultivation: after all explants were transferred to Erlenmeyer flasks, a layer of sterile filter paper was laid on the pre-incubated plates. Pouring the strain into a sterile bottle containing the explant after the strain is induced, pouring out the heavy suspension after 10min of infection, clamping the explant on filter paper by using sterile forceps, and sucking out more strain around the explant.

Naturally airing for 5min, and then spreading in a preculture medium containing a layer of filter paper. Followed by co-cultivation in the dark at 26℃for two days.

The medium used for the dark culture was MS medium supplemented with 30g/L sucrose, 9g/L agar bar, 0.7 mg/L6-BA and 0.25mg/L IAA, pH 5.9.

Screening culture of resistant callus and resistant buds

After the co-culture, the explants were transferred to callus and shoot induction medium for culture. The composition of the callus and bud induction culture medium is MS culture medium, 30g/L sucrose+9 g/L agar bar+0.7 mg/L6-BA and 0.25mg/L IAA are added, 220mg/L timentin+15 mg/L hygromycin is also added, and the pH value is 5.9.

The culture condition is 25 ℃, the photoperiod is 16h light/8 h dark, the light intensity is 5000lx, and the culture medium is replaced once for 18 days, so that the culture medium can form callus and adventitious buds.

Elongation culture of E-resistant adventitious buds

When the adventitious bud length is about 1cm, transferring the explant into a bud elongation culture medium, wherein the bud elongation culture medium is MS culture medium, 30g/L sucrose, 9g/L agar strip, 0.8 mg/L6-BA, 0.25mg/L IAA, 0.5mg/LGA3 and 230mg/L timentin, 15mg/L hygromycin are added, and the pH value is 5.9.

The culture condition is 28 ℃, the photoperiod is 16h light/8 h darkness, the light intensity is 5000lx, and the culture medium is replaced once in 18 days.

F rooting of resistant adventitious buds

When the length of the adventitious bud is 2-3cm, the adventitious bud is reduced by using a sterile scissors or cut by using a sterile planing knife. And placing the cut adventitious buds into a rooting medium for culturing.

The rooting medium is 1/2MS medium, 15g/L sucrose+9 g/L agar strips+0.6 mg/LIAA, 225mg/L timentin+15 mg/L hygromycin and the pH value is 5.9.

Culture conditions: culturing in a tissue culture room with the light period of 16h light/8 h dark and the light intensity of 5000lx at 28 ℃.

PCR molecular detection of G genetic transformation seedlings

When the adventitious bud length is 6-8cm and the root system is 4-7, the aseptic seedling is subjected to seedling hardening and transplanting.

After the aseptic seedlings are trained to survive, DNA of a genetic transformation strain is extracted by using a DNAsecure novel plant genome DNA extraction kit (Tiangen), PCR amplification is carried out on 700bp GFP fragments by using the genomic DNA of the Laohanganese as a template, and positive genetic transformation seedlings are screened.

The genetic transformation rate of examples 4-6 was 86% or more.

The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the embodiment of the present invention in any way, but any simple modification, equivalent variation and modification of the above embodiment according to the technical substance of the embodiment of the present invention still fall within the scope of the technical solution of the embodiment of the present invention.

Claims (9)

1. The regeneration method of the cantaloupe is characterized by comprising the following steps of:

(1) Seed sterilization and sterile explant acquisition: removing shells of the seeds of the Laohuan melon, sterilizing, culturing in a germination culture medium until the leaves of the Laohuan melon are completely unfolded and the true leaves are not grown, thus obtaining a sterile explant;

(2) Induction culture of callus: cutting off the head and tail of the sterile explant, inoculating a callus induction culture medium, and culturing for 15-20 days under the culture condition of 25-28 ℃;

(3) Induction culture of adventitious buds: after the callus appears, transferring the explant into a bud induction culture medium, and culturing until adventitious buds appear;

(4) Elongation of adventitious bud: when the length of the adventitious bud is 0.5-1cm, inoculating the explant with the adventitious bud into an adventitious bud elongation culture medium for culture;

(5) Induction culture of roots: when the adventitious buds are elongated to 1.5-3cm, cutting off the adventitious buds, and inoculating the adventitious buds into a root induction culture medium for culture.

2. The regeneration method according to claim 1, wherein,

in the step (1), the germination medium is: adding 15-20g/L sucrose and 8-9g/L agar strips into 1/2MS culture medium, and adjusting pH to 5.8-6.0;

in the steps (2) - (3), the culture medium is as follows: adding 30g/L sucrose, 9g/L agar, 0.5-1.0 mg/L6-BA, 0.15-0.3mg/L IAA into MS culture medium, and pH value is 5.8-6.0;

in the step (4), the adventitious bud elongation culture medium is: adding 30g/L sucrose, 9g/L agar, 0.5-1.0 mg/L6-BA, 0.15-0.3mg/L IAA, 0.5-1.0mg/LGA3 into MS culture medium, and pH value is 5.8-6.0;

in the step (5), the root induction culture medium is: 15g/L sucrose, 9g/L agar strips and 0.6-0.9mg/L IAA are added into the 1/2MS culture medium, and the pH value is 5.8-6.0.

3. The regeneration method according to claim 2, wherein,

in the step (1), the germination medium is: 15g/L sucrose and 8g/L agar strips are added into a 1/2MS culture medium, and the pH value is 5.8;

in the steps (2) - (3), the culture medium is as follows: 30g/L sucrose, 9g/L agar strips, 1.0 mg/L6-BA and 0.2mg/L IAA are added into an MS culture medium;

in the step (4), the adventitious bud elongation culture medium is: 30g/L sucrose, 9g/L agar, 1.0 mg/L6-BA, 0.3mg/L IAA and 1.0mg/LGA3 are added into the MS culture medium;

in the step (5), the root induction culture medium is: 1/2MS medium was supplemented with 15g/L sucrose, 9g/L agar strips, 0.8mg/L IAA.

4. The genetic transformation method of the cantaloupe is characterized by comprising the following steps of: the identification of genetically transformed seedlings after the regeneration method according to any one of claims 1 to 3;

the method comprises the steps of (1) performing pretreatment, infection and co-culture of explants before the callus induction culture in the step (2), and then performing the callus induction culture;

the pre-culture process of the explant comprises the following steps: cutting off the head and tail of the sterile explant, inoculating the sterile explant into a preculture medium, and performing dark culture under the culture condition of 25-28 ℃;

the infection and co-culture process comprises the following steps: infecting the precultured Laohuan melon cotyledon with agrobacterium tumefaciens bacteria liquid carrying plant expression vector, transferring to callus inducing culture medium, separating the explant from the culture medium with sterile filter paper, and co-culturing;

in the induction culture of the adventitious buds in the step (3): the adventitious bud induction culture also comprises timentin and an antibiotic hygromycin B;

elongation of adventitious buds in the step (4): the adventitious bud elongation culture medium also comprises timentin and antibiotics hygromycin B;

the induction culture of the root in the step (5): the root induction culture medium also comprises timentin and the antibiotic hygromycin B;

identification of the genetically transformed seedlings: GFP fluorescence detection is carried out on the genetically transformed Laohuan seedlings after the induction culture of the roots; extracting genome DNA of genetically transformed Laohanganese melon seedling, PCR positive detection and screening positive genetically transformed seedling.

5. The genetic transformation method according to claim 4, wherein the genetic transformation method comprises the steps of,

in the step (2), the agrobacterium tumefaciens bacterial liquid is formed by resuspending agrobacterium tumefaciens carrying a plant expression vector in an MS liquid culture medium, wherein the MS liquid culture medium comprises the following components: MS basic powder, 15g/L sucrose, 15g/L glucose, pH value is 5.8;

the culture medium for the preculture is MS culture medium, 30g/L sucrose, 9g/L agar, 0.25mg/L6-BA and 0.15mg/L IAA;

in the step (3), the concentration of the timentin is 200-250mg/L, and the concentration of the hygromycin is 10-20mg/L;

in the step (4), the concentration of the timentin is 200-250mg/L, and the concentration of the hygromycin is 10-20mg/L;

in the step (5), the concentration of the timentin is 200-250mg/L, and the concentration of the hygromycin is 10-20mg/L.

6. The genetic transformation method according to claim 5, wherein the genetic transformation method comprises the steps of,

in the step (2), the agrobacterium tumefaciens bacterial liquid also contains 50-100mg/L acetosyringone.

7. The genetic transformation method according to claim 5, wherein the genetic transformation method comprises the steps of,

OD of the agrobacterium tumefaciens bacterial liquid ₆₀₀ 0.4-0.6.

8. The genetic transformation method according to claim 4, wherein the genetic transformation method comprises the steps of,

the agrobacterium tumefaciens is agrobacterium tumefaciens GV3101, and the plant expression vector is pCAMBIA1302.

9. The genetic transformation method according to claim 4, wherein the genetic transformation method comprises the steps of,

in the step (2), the infection and co-culture process is carried out for 2 days in dark under the culture condition of 25-28 ℃;

in the step (3), culturing is carried out in a tissue culture chamber with the photoperiod of 16h illumination/8 h darkness and the illumination intensity of 5000lx at the temperature of 25-28 ℃ and the culture medium is replaced for 15-20 days;

in the step (4), culturing is carried out in a tissue culture chamber with the photoperiod of 16h illumination/8 h darkness and the illumination intensity of 5000lx at the temperature of 25-28 ℃ and the culture medium is replaced for 15-20 days;

in the step (5), culturing is carried out in a tissue culture room with the photoperiod of 16h illumination/8 h darkness and the illumination intensity of 5000lx at the temperature of 25-28 ℃.