CN114836463A

CN114836463A - Efficient genetic transformation method for broccoli by taking fleshy pedicel as explant

Info

Publication number: CN114836463A
Application number: CN202210311643.2A
Authority: CN
Inventors: 虞慧芳; 顾宏辉; 盛小光; 王建升; 沈钰森; 赵振卿
Original assignee: Zhejiang Academy of Agricultural Sciences
Current assignee: Zhejiang Academy of Agricultural Sciences
Priority date: 2022-03-28
Filing date: 2022-03-28
Publication date: 2022-08-02
Anticipated expiration: 2042-03-28
Also published as: CN114836463B

Abstract

The invention discloses a method for efficiently genetically transforming broccoli by taking fleshy pedicel as an explant, which comprises the following steps: (1) selecting a fleshy pedicel explant; (2) and (3) sterilizing the explants: cleaning and sterilizing the fleshy pedicel; (3) pre-culturing explants; (4) carrying out dip dyeing on the explants; (5) co-culturing the explant and the bacterial liquid; (6) and (3) delayed culture of explants: (7) screening and culturing explants; (8) rooting culture of the resistant bud; (9) positive detection of regenerated plants: and (3) detecting plants which are positive through PCR and GUS staining, and obtaining the broccoli genetic transformation positive seedlings. According to the method disclosed by the invention, the fleshy pedicel can regenerate and bud in a short time, the positive rate of the genetic transformation plant is 22-25%, the time required by transformation is short, the transformation result is stable, and the test repeatability is high.

Description

Efficient genetic transformation method for broccoli by taking fleshy pedicel as explant

Technical Field

The invention relates to a high-efficiency genetic transformation method for broccoli, in particular to a high-efficiency genetic transformation method for broccoli by taking fleshy pedicel as an explant.

Background

Broccoli (Brassica oleracea L.var. italica) is a variety of edible products in Brassica species of the brassicaceae family, and is highly popular among people because of rich nutrition and rich sulforaphane. As global warming, abnormal weather increasingly affects broccoli field production. Research on cultivation of broccoli varieties with disease and pest resistance, high temperature resistance, severe cold resistance, drought resistance and the like is very important for agricultural safety production. The agrobacterium-mediated genetic transformation technology is an important means for rapid transfer of exogenous excellent genes and site-directed mutation of target endogenous genes, and is also an important link in gene function verification. Therefore, the establishment of the efficient genetic transformation method for broccoli has important significance for molecular breeding and gene function research of broccoli.

At present, the genetic transformation success of broccoli is reported less, and the problems of low transformation efficiency, poor experiment repeatability and the like exist.

Disclosure of Invention

The invention aims to provide a high-efficiency genetic transformation method for broccoli by taking fleshy pedicel as an explant, which solves the problem of genetic transformation of broccoli, the fleshy pedicel can regenerate and bud in a short time, the positive rate of a genetic transformation plant is 22-25%, the transformation time is short, the transformation result is stable, and the test repeatability is high.

In order to achieve the above object, the present invention provides a method for efficient genetic transformation of broccoli using fleshy pedicel as explant, comprising:

(1) selecting a fleshy pedicel explant: removing upper buds of flower bulbs of mature broccoli, and selecting fleshy pedicel with the transverse diameter of 0.3-1.8 cm as an explant; for the explant, if the cross diameter of the explant is less than 0.3cm, the explant is too tender and weak in tolerance capacity to agrobacterium, and the contact area between the incision and the staining solution is too small, so that the genetic transformation positive rate is remarkably reduced; the transverse diameter of the explant is larger than 1.8cm, the incision area is too large, a large amount of quinone substances can be generated in the culture process, incision browning is accelerated, and the bud regeneration efficiency is obviously reduced. Therefore, the pedicel explant with the transverse diameter of 0.3-1.8 cm has higher bud regeneration capacity and stronger tolerance to agrobacterium simultaneously (when the agrobacterium is impregnated for 20-30 min, the browning degree of the explant is very low), and is most suitable for agrobacterium-mediated genetic transformation;

(2) and (3) sterilizing the explants: cleaning and sterilizing the fleshy pedicel;

(3) pre-culturing explants: sucking water on the surface of the sterilized fleshy pedicel, cutting wounds at two ends of the fleshy pedicel, cutting the explant into small sections with the length of 0.5-1.5 cm, pre-culturing in a pre-culture medium for 3-7 days at 25 ℃, and lighting for 16 h/d; wherein the explant pre-culture medium comprises: MS culture medium, 30g/L sucrose, 8g/L agar, 1.5-2.0 mg/L6-BA, 0.01-0.05 mg/L NAA, 0.4-0.5 mg/LTrans-ZT, and the pH is 5.6-6.0;

(4) and (3) carrying out dip dyeing on explants: placing the pre-cultured explant in an explant dip-dyeing solution for dip-dyeing; wherein, the preparation of the explant staining solution comprises the following steps: the agrobacterium strain GV3101 for constructing the target gene vector pCambia1301 is shake-cultured at 28 ℃ overnight to the bacterial liquid OD ₆₀₀ Centrifuging the bacterial liquid, discarding the supernatant, suspending the bacterial liquid by using sterile MS-sucrose solution with the same volume, and adding acetosyringone with the final concentration of 20mg/L to obtain an explant staining solution; wherein the sterile MS-sucrose solution comprises: MS liquid culture medium and 20g/L sucrose; the nucleotide sequence of the target gene is shown as SEQ ID NO. 1;

(5) co-culturing explants and bacterial liquid: sucking up liquid on the surface of the impregnated explant, adding a piece of sterile filter paper on the surface layer of the explant co-culture medium, placing the explant on the filter paper after the filter paper is wetted, and co-culturing for 3d under the dark condition; wherein the explant co-culture medium comprises: MS culture medium, 30g/L sucrose, 9g/L agar, 1.5-2.0 mg/L6-BA, 0.01-0.05 mg/LNAA, 0.4-0.5 mg/L Trans-ZT, and pH is 5.6-6.0; sterile filter paper is paved on the surface layer of a co-culture medium, so that the pollution rate of the explant can be effectively reduced, the co-culture stage is an important stage of agrobacterium infection of incision cells of the explant, the activity of the agrobacterium in a dry environment can be greatly reduced and even die, the high humidity is favorable for maintaining the activity of the agrobacterium, and the infection positive rate is improved, so that the concentration of agar in the co-culture medium is smaller than that of other types of culture media, and the water content in the culture media is larger; the invention can carry out agrobacterium infection for a longer time (3d), but does not damage explants, and can improve the positive rate of regeneration plants;

(6) and (3) delayed culture of explants: transferring the explants subjected to co-culture to an explant delay culture medium, and performing delay culture at 25 ℃ for 5-7 d with illumination time of 16 h/d; wherein the explant delay medium comprises: MS culture medium, 30g/L sucrose, 9g/L agar, 1.5-2.0 mg/L6-BA, 0.01-0.05 mg/LNAA, 0.4-0.5 mg/LTrans-ZT and 300mg/L timentin, wherein the pH value is 5.6-6.0;

(7) screening and culturing explants: transferring the explants subjected to delayed culture to an explant screening culture medium, carrying out screening culture at 25 ℃, wherein the illumination time is 16h/d, and transferring the explants to a new explant screening culture medium every 10-15 d until resistant buds grow; wherein the explant screening medium comprises: MS culture medium, 30g/L sucrose, 8g/L agar, 1.5-2 mg/L6-BA, 0.01-0.05 mg/L LNAA, 300mg/L timentin and 5-50 mg/L hygromycin, and the pH value is 5.6-6.0;

(8) rooting culture of the resistant buds: cutting the regenerated resistant bud from the lower part of the growing point, transferring the cut regenerated resistant bud into a bud rooting culture medium, and culturing until a root grows; wherein the shoot rooting medium comprises: MS culture medium, 0.05-0.1 mg/LNAA, 30g/L sucrose, 8g/L agar, 300mg/L timentin and 5mg/L hygromycin, and the pH value is 5.6-6.0;

(9) positive detection of regenerated plants: performing double detection through PCR and GUS staining to obtain positive plants, which are genetically transformed positive seedlings of broccoli; wherein, in the PCR, genome DNA of resistant seedling leaves after rooting is taken as a template, nucleotide sequences of adopted primers are shown as SEQ ID NO.6 and SEQ ID NO.7, and a result is positive if a strip of a PCR product is at 658 bp; and (4) carrying out GUS staining, wherein a blue dot is arranged on a white background of the leaf or the blue is positive.

Preferably, the broccoli is selected from plants and healthy broccoli growing in flower bulbs.

Preferably, the fleshy pedicel is sterilized by using a 2-4% sodium hypochlorite solution.

Preferably, the fleshy pedicel is sterilized: and (3) cutting the fleshy pedicel into small sections within 5cm, sterilizing by adopting a 2-4% sodium hypochlorite solution, and cleaning by adopting sterile water.

Preferably, the sterilization time of the fleshy pedicel is 10-15 min.

Preferably, the explant is subjected to dip dyeing, and the dip dyeing time is 10-30 min.

Preferably, the PCR reaction system is: template, 2 XPCRmix, upstream primer, downstream primer and water.

Preferably, the PCR reaction procedure is: 94 ℃ for 3 min; 30 cycles of 94 ℃ for 30s,55 ℃ for 15s,72 ℃ for 15 s; extension at 72 ℃ for 7min and holding at 16 ℃.

Preferably, the GUS staining is: taking leaves of PCR detection positive plants, adding GUS staining working solution to completely immerse the leaves, placing the leaves in a vacuum-pumping tank for vacuum-pumping for 5min, and then preserving heat at 30 ℃ for 2 h; transferring the leaves into 70% ethanol for decolorization for 3-4 times until the negative control is white, wherein the negative control is the leaves of plants without 658bp bands detected by PCR; the positive plants are observed by naked eyes with blue dots on the white background of the leaves.

The efficient genetic transformation method of broccoli by taking fleshy pedicel as explant solves the problem of genetic transformation of broccoli, and has the following advantages:

the invention establishes a high-efficiency genetic transformation system taking the fleshy pedicel of broccoli as an explant for the first time, and combines PCR detection and GUS dyeing results, so that the positive rate of a genetic transformation plant is about 22-25% (which is better than the reported time and transformation efficiency required by broccoli genetic transformation). The method can realize regeneration of the transgenic positive seedlings within 2-3 months, has short transformation time, stable transformation result and high test repeatability, and has important significance for molecular breeding and gene function research of broccoli.

Drawings

FIG. 1 is a photograph of fleshy cauliflower regenerated shoots obtained by the method of the present invention.

FIG. 2 shows the results of the detection of the regenerated resistant seedlings (A) and non-resistant seedlings (B) obtained by the method of the present invention.

FIG. 3 shows the PCR detection (A) and GUS staining (B) results of the target gene of the cauliflower fleshy pedicel regeneration resistant plant by the method of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

1. Preparation of culture Medium

The medium comprises the components of the medium at each stage of genetic transformation, which are described in the following formula. Note: the basic culture medium (MS + sucrose + agar) is prepared and then is sterilized under high pressure for 20min at 121 ℃, and various hormones, timentin and hygromycin are cooled to about 60 ℃ after being sterilized at high temperature in the basic culture medium and then are added.

(1) Explant pre-culture medium: MS culture medium + sucrose 30g/L + agar 8g/L +6-BA 1.5 mg/L + NAA0.01 mg/L + Trans-ZT 0.4mg/L (Trans-zeatin, CAS number: 1637-39-4, purchased from Mecanne), pH 5.8;

(2) explant co-culture medium: MS culture medium, sucrose 30g/L, agar 9g/L, 6-BA 1.5 mg/L, NAA0.01 mg/L, Trans-ZT 0.4mg/L and pH 5.8;

(3) explant delay medium: MS culture medium, sucrose 30g/L, agar 9g/L, 6-BA 1.5 mg/L, NAA0.01 mg/L, Trans-ZT 0.4mg/L, timentin 300mg/L and pH 5.8;

(4) explant screening medium: MS culture medium, 30g/L of cane sugar, 8g/L of agar, 1.5 mg/L of 6-BA, 0.01mg/L of NAA, 300mg/L of timentin and 50mg/L of hygromycin, and the pH value is 5.8;

(5) bud rooting culture medium: MS culture medium + NAA 0.05mg/L + sucrose 30g/L + agar 8g/L + timentin 300mg/L + hygromycin 5mg/L, pH 5.8.

2. Construction of Broccoli Gene expression vector

(1) Cloning of genes

Primers were designed based on the TO1000SOC1 gene sequence (SEQ ID NO.1) and the sequences were as follows:

SOC1-KpnI-F(SEQ ID NO.2)：

GCGGGTCGACGGTACCATGGTGAGGGGGAAAACTC；

SOC1-KpnI-R(SEQ ID NO.3)：

TAGACATATGGGTACCTCACTTTCTTGAAGAACAAGG。

RNA was extracted from broccoli shoots and reverse-transcribed into cDNA (using the HiScript 1st Strand cDNA Synthesis Kit from Vazyme) by the following procedures:

(1.1) denaturation of RNA template

A mixture as shown in Table 1 below was prepared in RNase-free centrifuge tubes, heated at 65 ℃ for 5min, rapidly chilled on ice, and allowed to stand on ice for 2 min.

TABLE 1 shows the components of the mixture for denaturation of RNA template

(1.2) first Strand cDNA Synthesis

And (3) mixing the mixed solution obtained in the step (1.1) with 2 XRT Mix and HiScript II Enzyme Mix, specifically shown in Table 2, gently blowing and stirring the mixture by using a pipette, reacting at 50 ℃ for 45min, and reacting at 85 ℃ for 5 min.

Table 2 shows the composition of the mixture for cDNA Synthesis

(1.3) PCR amplification

PCR was performed using the above primers SOC1-KpnI-F and SOC1-KpnI-R using cDNA as a template (phanta max super-fidelity DNA Polymerase from Vazyme).

The PCR reaction system (total volume 50. mu.L) is shown in Table 3 below:

the PCR reaction program is: 30s at 95 ℃; (95 ℃ for 15s, annealing temperature for 55 ℃ for 15s, extension for 72 ℃ for 1min) x 39 cycles; finally, the extension is carried out at 72 ℃ for 5 min.

The PCR product was subjected to gel electrophoresis, and the result showed that a single band (642bp) was amplified and the size was correct.

(2) Vector construction

The vector pCambia1301(VT1842, from Youbao, the structure of which is shown in FIG. 3) was linearized by a single cleavage with KpnI (using the corresponding restriction enzyme products from Thermo or Takara), and the cleavage reaction systems are shown in Table 4 below.

Table 4 shows the digestion reaction system

The digested products were purified and then subjected to recombination reaction with the PCR products (recombination reaction Kit: Clonexpress-II One Step Cloning Kit from Vazyme), and the recombination ligation reaction system (total volume 10. mu.l) was as shown in Table 5 below.

Table 5 shows the recombination ligation reaction system

And (3) lightly sucking and uniformly mixing the reaction liquid by using a pipette, centrifuging for a short time to collect the reaction liquid to the bottom of the tube, placing the tube at 37 ℃ for reaction for 30min, and immediately placing the tube on ice for cooling.

(3) Recombinant product transformed Escherichia coli DH5 alpha cell

The specific transformation procedure for transforming E.coli DH5 alpha cells with the recombinant product is as follows:

(3.1) adding 10. mu.L of the product of the above recombinant ligation reaction to 100. mu.L of E.coli competent cells;

(3.2) carrying out ice bath for 30 min;

(3.3) carrying out heat shock at 42 ℃ for 60-90 s;

(3.4) carrying out ice bath for 2 min;

(3.5) adding 800. mu.L of LB liquid medium;

(3.6) shaking-culturing at 37 ℃ for 30 min;

(3.7) centrifuging at 6000rpm for 3min, discarding the supernatant, and spreading Kana (50mg/L) resistant culture medium plates;

(3.8) carrying out inverted culture at 37 ℃ for 12-16h, and then picking resistant colonies;

(3.9) adding 100. mu.L of LB (containing Kana) liquid medium to each well of a 96-well plate;

(3.10) taking 4-8 colonies from each plate, and carrying out amplification culture at 37 ℃ and 180rpm for 2 h;

(3.11) taking 1 mu L of bacterial liquid to carry out PCR positive detection;

(3.12) selecting PCR positive transformant shake bacteria, culturing and extracting plasmids, sequencing the amplified product, wherein primers for amplification and sequencing are vector sequences inserted into two sides of a target gene and comprise:

35S-F(SEQ ID NO.4)：

5’-GACGCACAATCCCACTATCC-3’；

2301-F(SEQ ID NO.5)：

5’-GCTTCCGGCTCGTATGTTG-3’。

(4) positive plasmid transformed Agrobacterium competent cell (GV3101)

After sequencing the PCR amplification product, positive plasmids containing target genes are transformed into agrobacterium GV3101 competent cells, and the specific transformation steps are as follows:

(4.1) thawing 100. mu.L of Agrobacterium GV3101 competent cells on ice;

(4.2) adding 3. mu.L of pCambia1301 plasmid DNA with the target fragment, standing on ice for 30min, freezing in liquid nitrogen for 1min, and then water-bathing at 37 ℃ for 3 min;

(4.3) adding 900 mu L of YEP culture medium without antibiotics, carrying out shake culture at 28 ℃ and 200rpm/min for 3 h;

(4.4) centrifuging at 10000rpm for 1min to concentrate the bacterial liquid, and redissolving the bacterial cells with 100 mu LYEP;

(4.5) the cells after the redissolution were applied to a solid YEP medium supplemented with 50mg/L kanamycin and cultured at 28 ℃ for 12 to 16 hours.

(4.6) PCR identifies positive colonies, and the PCR primers are as follows: 35S-F and 2301-F.

(5) Analysis of vector sequencing results

And (3) comparing the sequencing results, and displaying the results: the target gene is inserted into the vector, and the construction of the expression vector is accurate.

3. Agrobacterium-mediated broccoli genetic transformation regeneration plant obtaining method

(1) Selection of fleshy pedicel explants

Selecting healthy broccoli growing on a plant and a flower ball as donor plants, taking the flower ball with the diameter of about 10cm, removing upper flower buds, and selecting a fleshy peduncle with the transverse diameter of 0.3-1.8 cm as an explant.

(2) Sterilization of explants

The fleshy pedicel is cut into small sections within 5cm, and is convenient to disinfect and clean, the small sections are cleaned by purified water for 3 times, then the small sections are put into a 2% sodium hypochlorite solution, the surface of the small sections is disinfected for 15min by slight shaking (60rpm) of a shaking table, and then the small sections are cleaned by sterile water for 4 times on an ultra-clean bench for later use.

(3) Pre-culture of explants

Placing the sterilized fleshy pedicel on sterile filter paper on a super clean bench, sucking surface water, and cutting wounds at two ends of the fleshy pedicel. Finally, the explants were cut into small pieces of about 0.5-1.5 cm in length and pre-cultured in an explant pre-culture medium for 3 days (25 ℃, 16h/d light).

(4) Preparation of explant staining solution

Agrobacterium strain GV3101, which had been constructed with the vector (pCambia1301) of the target gene SOC1(SEQ ID NO.1), was shake-cultured at 28 ℃ overnight to the bacterial liquid OD ₆₀₀ ＝1.0. Centrifuging the bacterial liquid at 5000rmp for 10min, and removing the supernatant; suspending with sterile MS-sucrose solution (MS liquid culture medium +20g/L sucrose, high temperature sterilization) with the same volume, adding acetosyringone with the final concentration of 20mg/L to obtain explant staining solution, and standing at normal temperature for 1h for later use.

(5) Dip staining of explants

And (4) lightly placing the explants which are pre-cultured for 3d and have no pollution into the bacterial liquid prepared in the step (4) for 10min by using sterile long-handled tweezers.

(6) Co-culturing explant with bacterial liquid

And lightly placing the impregnated explant on sterile filter paper by using a sterile long-handle forceps, and sucking the surface bacterial liquid. Adding a piece of sterile filter paper to the surface layer of the explant co-culture medium, vertically placing the explant on the filter paper after the filter paper is wetted, and co-culturing for 3d under the dark condition. The placed sterile filter paper can absorb the residual agrobacterium after the infection of the explant, and the pollution rate of the explant is reduced.

(7) Delayed explant culture

The explants after 3d of co-cultivation are transferred to an explant delay medium and delayed for 7d (25 ℃, 16h/d of light).

(8) Explant screening culture

After the delayed culture is finished, the explants are transferred to an explant screening medium (25 ℃, the light is 16h/d), and the explants are transferred to a new explant screening medium every 15d until resistant buds grow out (about 20 days). Finally, a total of 121 uncontaminated explants were obtained, of which 69 regenerated resistant shoots.

(9) Rooting culture of resistant buds

And cutting the regeneration bud from the lower part of the growing point, transferring the regeneration bud into a bud rooting culture medium, culturing until roots grow, and finally obtaining 88 resistant plants with good rooting.

4. Positive detection of regenerated plants

(1) PCR detection of resistant regenerated plants

After the target gene (SOC1) is constructed into the vector (pCambia1301), PCR primers are designed according to the target gene and the vector sequence, and the primer sequences are as follows:

upstream primer SOC1TransPF1(SEQ ID NO. 6):

GCGGGTCGACGGTACC(5’-3’)；

downstream primer SOC1TransPR1(SEQ ID NO. 7):

TCACTTTCTTGAAGAACAAGGTAACC(5’-3’)。

and (3) performing PCR amplification by using the genome DNA of the resistance seedling leaves after rooting as a template, the DNA of untransformed plant leaves as a negative control and the recombinant plasmid pCambia1301 as a positive control.

The PCR procedure was: 94 ℃ for 3 min; 30 cycles of 94 ℃ for 30s,55 ℃ for 15s,72 ℃ for 15 s; extension at 72 ℃ for 7min and holding at 16 ℃.

The PCR reaction system is as follows: 1 μ L template, 10 μ L2 XPCR mix, 1 μ L10 μ M forward primer, 1 μ L10 μ M reverse primer and 7 μ L water.

And after the PCR reaction is finished, performing 1% agarose gel electrophoresis on the PCR product, wherein a negative control has no target band, a positive control has a bright target band (658bp, SEQ ID NO.8), the transformed resistant seedling leaf genome DNA has a 658bp band which is a positive plant, and the non-banded plant is an untransformed plant. The results showed that of 88 resistant plants with good roots, 30 plants with a 658bp band were PCR-detected.

(2) GUS staining for PCR detection of positive plant leaves

GUS staining solutions were prepared according to the instructions of the GUS staining solution kit (purchased from Leagene, Beijing, under CAS number 18656-96-7).

Cutting young leaves of 88 PCR detection positive plants into a vial, adding a proper amount of GUS staining working solution to completely immerse the young leaves, placing the young leaves in a vacuum-pumping tank, vacuumizing for 5min, and then preserving heat at 30 ℃ for 2 h; transferring the leaves into 70% ethanol for decolorization for 3-4 times until negative control (untransformed plant leaves) is white; and (4) observing by naked eyes, wherein a blue dot (GUS expression site) is arranged on the white background of the leaf, and the positive plant is obtained. The results showed that there were 27 plants with blue dots stained with GUS among 30 plants with the band of interest detected by PCR.

27 broccoli genetic transformation positive seedlings (PCR detection and GUS staining detection are positive seedlings with successful genetic transformation) are obtained in a short time by using the method, 121 uncontaminated explants are used for experiments in total, and the genetic transformation efficiency is 27/121 x 100 percent to 22.31 percent.

Example 2

1. Preparation of culture Medium

Essentially the same as in example 1, except for the concentrations of the components, the medium formulation at each stage of the genetic transformation in this example was as follows:

(1) explant pre-culture medium: MS culture medium, sucrose 30g/L, agar 8g/L, 6-BA 2.0mg/L, NAA 0.05mg/L, Trans-ZT 0.5mg/L and pH 6.0;

(2) explant co-culture medium: MS culture medium, sucrose 30g/L, agar 9g/L, 6-BA 2.0mg/L, NAA 0.05mg/L, Trans-ZT 0.5mg/L and pH 6.0;

(3) explant delay medium: MS culture medium, sucrose 30g/L, agar 9g/L, 6-BA 2.0mg/L, NAA 0.05mg/L, Trans-ZT 0.5mg/L, and timentin 300mg/L, and pH is 6.0;

(4) explant screening medium: MS culture medium, sucrose 30g/L, agar 8g/L, 6-BA 2.0mg/L, NAA 0.05mg/L, Trans-ZT 0.5mg/L, timentin 300mg/L and hygromycin 5mg/L, and pH is 6.0;

(5) bud rooting culture medium: MS culture medium + NAA 0.1mg/L + sucrose 30g/L + agar 8g/L + timentin 300mg/L + hygromycin 5mg/L, pH 6.0;

2. agrobacterium-mediated broccoli genetic transformation regeneration plant obtaining method

(1) Selection of fleshy pedicel explants

Selecting healthy broccoli growing plants and flower bulbs as donor plants, and taking the flower bulbs with the diameter of about 12 cm. Removing upper buds, and selecting fleshy pedicel with transverse diameter of 0.3-2.0cm as explant.

(2) Sterilization of explants

Cutting fleshy pedicel into small segments within 5cm, conveniently disinfecting and cleaning, cleaning with purified water for 3 times, then placing into 4% sodium hypochlorite solution, slightly shaking (60rpm) in a shaking table for surface disinfection for 10min, and cleaning with sterile water for 4 times on an ultra-clean bench for later use;

(3) pre-culture of explants

Placing the sterilized fleshy pedicel on sterile filter paper on a super clean bench, sucking surface water, and cutting the wound soaked by the sterilization liquid at two ends of the fleshy pedicel. Finally, the explants were cut into small pieces of about 0.5-1.5 cm in length and pre-cultured in the explant pre-culture medium for 7 days (25 ℃, 16h/d light).

(4) Preparation of explant staining solution

The Agrobacterium strain GV3101 with the constructed target gene vector (pCambia1301) was shake-cultured overnight at 28 ℃ until the OD of the bacterial liquid ₆₀₀ 0.6. Centrifuging the bacterium solution at 5000rmp for 10 minutes, and removing the supernatant; suspending with sterile MS-sucrose solution (MS liquid culture medium +20g/L sucrose, high temperature sterilization) with the same volume, adding acetosyringone with final concentration of 20mg/L, standing at room temperature for 1h, and keeping.

(5) Dip-staining of explants

And (3) slightly putting the explants which are pre-cultured for 7 days and have no pollution into the bacteria solution for dip-dyeing for 30 minutes by using sterile long-handled tweezers.

(6) Co-culturing explant with bacterial liquid

And lightly placing the impregnated explant on sterile filter paper by using a sterile long-handle forceps, and sucking the surface bacterial liquid. Adding a piece of sterile filter paper to the surface layer of the co-culture medium, vertically placing the explant on the filter paper after the filter paper is wetted, and co-culturing for 3d under the dark condition.

(7) Delayed explant culture

After 3d of co-cultivation, the explants were transferred to explant delay medium and delayed for 5d (25 ℃, 16h/d of light).

(8) Explant screening culture

After the delayed culture was completed, the explants were transferred to explant selection medium and selection cultured for 4 weeks (25 ℃, light 16h/d), wherein the explants were transferred to new explant selection medium every 10d until resistant shoots developed. Finally, a total of 113 uncontaminated explants were obtained, of which 72 regenerated resistant shoots.

(9) Rooting culture of resistant buds

And cutting the regenerated resistant buds from the lower part of the growing point, transferring the cut regenerated resistant buds to a bud rooting culture medium, culturing until roots grow, and finally obtaining the resistant plants with good rooting, wherein the total number of the resistant plants is 80.

3. Positive detection of regenerated plants

(1) PCR detection of resistant regenerated plants

The same method as that of example 1 was used for detection, and the results showed that: among 80 resistant plants with good rooting, 33 plants with 658bp target band were detected by PCR.

(2) GUS staining for PCR detection of positive plant leaves

The same method as that of example 1 was used for detection, and the results showed that: PCR was performed to detect 29 plants with blue spots in GUS staining among 33 plants with the objective band.

29 strains of genetic transformation positive seedlings of broccoli (PCR and GUS staining are positive seedlings which are successful in genetic transformation) are obtained in a short time by using the method, the total number of non-polluted explants used for experiments is 113, and the genetic transformation efficiency is 29/113-25.66%.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

Comparative example 1

The procedure for genetic transformation of regenerated plants with broccoli as in example 1 was followed, except that the concentrations of 6-BA, NAA and Trans-ZT in the medium were varied.

TABLE 6 Effect of different concentrations of 6-BA and NAA combinations on the Broccoli fleshy peduncle regenerating efficiency

TABLE 7 influence of different concentrations of Trans-ZT added on the regeneration bud efficiency and time of fleshy pedicel of broccoli

6-BA is an artificial chemically synthesized cytokinin to promote bud regeneration, while NAA is an auxin to promote root growth. The invention researches the combination of cytokinin and auxin with different concentrations, and the germination rates of cauliflower peduncle explants are completely different under different concentration ratios. In addition, by adding Trans-ZT (Trans-zeatin, natural cytokinin) with a certain concentration, the regeneration of the buds of the fleshy pedicel explant can be induced more quickly, the bud regeneration time is shortened, and the high-frequency bud regeneration effect is maintained.

Sequence listing

<110> Zhejiang province academy of agricultural sciences

<120> efficient genetic transformation method of broccoli by taking fleshy pedicel as explant

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 642

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggtgaggg ggaaaactca gatgaagcga atagagaatg caacaagcag acaagtgact 60

ttctctaagc gaaggaatgg tttgttgaaa aaagcctttg agctctcagt gctttgtgat 120

gctgaagttt ctctgatcat cttctctcct aaggcaaaac tttatgaatt cgccagctcc 180

aatatgcaag ataccataga tcgttatctg aggcatacca aggatcgtgt cagcaccaaa 240

cctgtttctg aagaaaattt gcagcatttg aaacatgagg cagcaaacat gatgaagaaa 300

attgaacaac tcgaagcttc caaacgtaaa ctcttgggag aaggcatagg atcatgttcg 360

atagaggagc tgcagcaaat tgagcaacaa cttgagaaaa gtgtcaaatg tatccgagct 420

agaaagactc aagtgtttaa ggaacaaatt gagcagctca agcaaaagga gaaagctcta 480

gctgcagaaa acaagaagct cgctgaaaag tggggatctc atgaaatcga agtccggtcg 540

aataagaacc aagaaagtgg aagaggtgac gaagagagta gcccaagttc tgaagtagag 600

acagagttgt tcattgggtt accttgttct tcaagaaagt ga 642

<210> 2

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gcgggtcgac ggtaccatgg tgagggggaa aactc 35

<210> 3

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tagacatatg ggtacctcac tttcttgaag aacaagg 37

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gacgcacaat cccactatcc 20

<210> 5

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gcttccggct cgtatgttg 19

<210> 6

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gcgggtcgac ggtacc 16

<210> 7

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tcactttctt gaagaacaag gtaacc 26

<210> 8

<211> 658

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gcgggtcgac ggtaccatgg tgagggggaa aactcagatg aagcgaatag agaatgcaac 60

aagcagacaa gtgactttct ctaagcgaag gaatggtttg ttgaaaaaag cctttgagct 120

ctcagtgctt tgtgatgctg aagtttctct gatcatcttc tctcctaagg caaaacttta 180

tgaattcgcc agctccaata tgcaagatac catagatcgt tatctgaggc ataccaagga 240

tcgtgtcagc accaaacctg tttctgaaga aaatttgcag catttgaaac atgaggcagc 300

aaacatgatg aagaaaattg aacaactcga agcttccaaa cgtaaactct tgggagaagg 360

cataggatca tgttcgatag aggagctgca gcaaattgag caacaacttg agaaaagtgt 420

caaatgtatc cgagctagaa agactcaagt gtttaaggaa caaattgagc agctcaagca 480

aaaggagaaa gctctagctg cagaaaacaa gaagctcgct gaaaagtggg gatctcatga 540

aatcgaagtc cggtcgaata agaaccaaga aagtggaaga ggtgacgaag agagtagccc 600

aagttctgaa gtagagacag agttgttcat tgggttacct tgttcttcaa gaaagtga 658

Claims

1. A method for efficiently genetically transforming broccoli by taking fleshy pedicel as an explant is characterized by comprising the following steps:

(1) selecting a fleshy pedicel explant: removing upper buds of flower bulbs of mature broccoli, and selecting fleshy pedicel with the transverse diameter of 0.3-1.8 cm as an explant;

(3) pre-culturing explants: sucking water on the surface of the sterilized fleshy pedicel, cutting wounds at two ends of the fleshy pedicel, cutting the explant into small sections with the length of 0.5-1.5 cm, pre-culturing in a pre-culture medium for 3-7 days at 25 ℃, and lighting for 16 h/d; wherein the explant pre-culture medium comprises: MS culture medium, 30g/L sucrose, 8g/L agar, 1.5-2.0 mg/L6-BA, 0.01-0.05 mg/LNAA, 0.4-0.5 mg/LTrans-ZT, and the pH is 5.6-6.0;

(5) co-culturing explants and bacterial liquid: sucking up liquid on the surface of the impregnated explant, adding a piece of sterile filter paper on the surface layer of the explant co-culture medium, placing the explant on the filter paper after the filter paper is wetted, and co-culturing for 3d under the dark condition; wherein the explant co-culture medium comprises: MS culture medium, 30g/L sucrose, 9g/L agar, 1.5-2.0 mg/L6-BA, 0.01-0.05 mg/LNAA, 0.4-0.5 mg/LTrans-ZT, and the pH is 5.6-6.0;

(6) and (3) delayed culture of explants: transferring the explants subjected to co-culture to an explant delay culture medium, and performing delay culture at 25 ℃ for 5-7 d with illumination time of 16 h/d; wherein the explant delay medium comprises: MS culture medium, 30g/L sucrose, 9g/L agar, 1.5-2.0 mg/L6-BA, 0.01-0.05 mg/L NAA, 0.4-0.5 mg/L LTrans-ZT and 300mg/L timentin, wherein the pH value is 5.6-6.0;

(7) screening and culturing explants: transferring the explants subjected to delayed culture to an explant screening culture medium, carrying out screening culture at 25 ℃, wherein the illumination time is 16h/d, and transferring the explants to a new explant screening culture medium every 10-15 d until resistant buds grow; wherein the explant screening medium comprises: MS culture medium, 30g/L sucrose, 8g/L agar, 1.5-2 mg/L6-BA, 0.01-0.05 mg/L NAA, 300mg/L timentin and 5-50 mg/L hygromycin, wherein the pH value is 5.6-6.0;

(9) positive detection of regenerated plants: performing PCR amplification and GUS staining double detection on plants which are positive, wherein the plants are broccoli genetic transformation positive seedlings; wherein, in the PCR, genome DNA of resistant seedling leaves after rooting is taken as a template, the adopted primer sequences are shown as SEQ ID NO.6 and SEQ ID NO.7, and the result is positive if the strip of the PCR product is 658 bp; and (4) carrying out GUS staining, wherein a blue dot is arranged on a white background of the leaf or the blue is positive.

2. The efficient genetic transformation method for broccoli by taking fleshy pedicel as explant according to claim 1, wherein healthy broccoli grows from selected plants and flower balls of broccoli.

3. The efficient genetic transformation method for broccoli by taking fleshy pedicel as explant according to claim 1, wherein the fleshy pedicel is sterilized by using 2-4% sodium hypochlorite solution.

4. The efficient genetic transformation method of broccoli using fleshy pedicel as explant according to claim 1, wherein said fleshy pedicel is sterilized: and (3) cutting the fleshy pedicel into small sections within 5cm, sterilizing by adopting a 2-4% sodium hypochlorite solution, and cleaning by adopting sterile water.

5. The efficient genetic transformation method of broccoli by taking fleshy pedicel as explant according to claim 1, wherein the fleshy pedicel sterilization time is 10-15 min.

6. The efficient genetic transformation method for broccoli by taking fleshy pedicel as explant according to claim 1, wherein the explant is subjected to dip dyeing for 10-30 min.

7. The efficient genetic transformation method for broccoli by taking fleshy pedicel as explant according to claim 1, wherein the PCR reaction system is as follows: template, 2 × PCR mix, upstream primer, downstream primer and water.

8. The efficient genetic transformation method for broccoli by taking fleshy pedicel as explant according to claim 1, wherein the PCR reaction program comprises the following steps: 94 ℃ for 3 min; 30 cycles of 94 ℃ for 30s,55 ℃ for 15s,72 ℃ for 15 s; extension at 72 ℃ for 7min and holding at 16 ℃.

9. The efficient genetic transformation method for broccoli by taking fleshy pedicel as explant according to claim 1, wherein GUS staining comprises the following steps: taking leaves of PCR detection positive plants, adding GUS staining working solution to completely immerse the leaves, placing the leaves in a vacuum-pumping tank for vacuum-pumping for 5min, and then preserving heat at 30 ℃ for 2 h; transferring the leaves into 70% ethanol for decolorization for 3-4 times until the negative control is white, wherein the negative control is the leaves of plants without 658bp bands detected by PCR; the plants are observed by naked eyes, wherein the white background of the leaves has small blue spots or the blue is positive.