CN115386591B - Molecular breeding method of single herba Cichorii - Google Patents

Abstract

The invention discloses a molecular breeding method for monosedum. The method uses the leaves, petioles and stems of sterile seedlings as transformation receptors, introduces the constructed plant expression vector containing the target gene into the genome of monosedum through the Agrobacterium tumefaciens-mediated method, and differentiates to form regenerated plants through the bud organogenesis pathway; during transformation bud induction, transformation bud elongation and rooting, antibiotics corresponding to the screening marker gene and at appropriate concentrations are added to screen transformants, and Southern hybridization, Northern hybridization and GUS staining are used to prove that the obtained resistant strains are all transformed strains. The entire operation process of the method of the invention takes 4 months, and 100 leaf explants are operated at one time, and about 11 transgenic strains of strains can be obtained on average. Each transgenic leaf can produce more than 20 buds after 6 weeks of culture, and the rooting rate of the regenerated plants is 100%, and the survival rate of the transgenic plants transplanted into the soil is 100%.

Description

A molecular breeding method for single-seat chicory

Technical Field

The invention belongs to the technical field of plant genetic engineering, and in particular relates to a molecular breeding method for rapidly obtaining a large number of transgenic single-seat chicory new germplasms.

Background technique

Metabriggsia ovalifolia W.T.Wang is a plant of the Gesneriaceae family and the genus Metabriggsia. It is a plant endemic to limestone forests at an altitude of 1,100 meters in Guangxi (Naipo, Jingxi and Huanjiang counties), Guizhou (Libo) and Yunnan. It has important scientific research value in the evolution of Gesneriaceae plants, the regulation mechanism of plant growth and development in special habitats, and the maintenance of ecological species diversity. It also has extremely high economic development value in the development and utilization of horticultural plant resources. However, due to its need for special habitats, the plant has been listed as a Class I protected plant in China and listed in the IUCN Red List of Threatened Species. Saving Metabriggsia ovalifolia resources is urgent.

Genetically modified biotechnology is undoubtedly one of the most rapidly applied major technologies in modern biotechnology. It has unique advantages in breeding excellent plant varieties, especially for plants that require special habitats to complete their life history. The application research of traditional hybridization, introduction and domestication, bud mutation, physical and chemical mutagenesis and other technologies on these plants is limited. Therefore, genetically modified technology plays an extremely important role in the molecular breeding of these plants.

There are no reports of research papers and patents on transgenic technology of single-seat chicory. Therefore, the development of molecular breeding technology of single-seat chicory has important scientific research and production significance.

Summary of the invention

The purpose of the present invention is to provide a molecular breeding method for rapidly obtaining a large number of transgenic single-seat lettuce in view of the deficiency that there is no transgenic technology for single-seat lettuce in the prior art. A large number of new germplasms of single-seat lettuce can be obtained within 4 months, and the method is used to create new germplasms of early-flowering single-seat lettuce, filling the gap in the genetic transformation breeding technology of single-seat lettuce at home and abroad, meeting the current urgent need for genetic transformation technology for endangered scientific mechanism research and ornamental resource development of single-seat lettuce, and having significant scientific, ecological, economic and social benefits in saving its germplasm resources, maintaining species diversity and sustainable development.

A molecular breeding method for single-seat chicory of the present invention comprises the following steps:

S1. Cultivation of explants for transformation: the explants are leaves, petioles or stems of regenerated plants of the monocotyledonous spatholobi; the regenerated plants are prepared by the following steps: sterilizing young leaves of the monocotyledonous spatholobi, cutting them into 0.5-0.8 cm long segments, inoculating them into bud induction medium, culturing them until green callus appears and then develops into buds, and when the buds are up to 1 cm high, cutting them out and transferring them to rooting medium for culturing, inducing rooting to obtain regenerated plants;

The bud induction medium is MS medium supplemented with 1 mg/L BA, 0.1 mg/L NAA, 30 g/L sucrose and 8 g/L agar, pH 5.8;

The rooting medium is 1/2MS medium supplemented with 30 g/L sucrose and 8 g/L agar, pH 5.8;

S2. Transformation to obtain resistant plants: soak the explants prepared in step S1 in a bacterial solution of Agrobacterium tumefaciens containing a plant expression vector carrying the target gene for 25-35 minutes, then take out the explants, absorb the excess bacterial solution and transfer the explants to a co-culture medium, culture in the dark for 3 days, then take out the co-cultured explants, rinse and transfer the explants to a resistance bud induction screening medium I, culture under light, and after the resistance buds grow, transfer the resistance buds to a fresh resistance bud induction medium II, and when the resistance buds are up to 1 cm, transfer them to a resistance bud rooting medium, culture under light, and allow the resistance buds to elongate and produce adventitious roots to obtain resistant plants;

The co-culture medium is MS medium supplemented with 1 mg/L BA, 0.1 mg/L NAA, 20 mg/L acetosyringone, 30 g/L sucrose and 8 g/L agar, pH 5.5;

The resistant bud induction screening medium I is MS medium supplemented with 1 mg/L BA, 0.1 mg/L NAA, 15-30 mg/L hygromycin, 500 mg/L cephalosporin, 30 g/L sucrose and 8 g/L agar, pH 5.8;

The resistant bud induction screening medium II is MS culture supplemented with 1 mg/L BA, 0.1 mg/L NAA, 15-30 mg/L hygromycin and 250 mg/L cephalosporin, 30 g/L sucrose and 8 g/L agar, pH 5.8;

The resistance rooting medium is 1/2MS medium supplemented with 25-30 mg/L hygromycin, 250 mg/L cephalosporin, 30 g/L sucrose and 8 g/L agar, pH 5.8;

S3. Molecular detection of resistant plants: Use the selection marker gene, reporter gene or target gene sequence on the plant expression vector to perform Southern hybridization to detect whether the gene has been transferred into the genome of the single-seat lettuce, use GUS staining or Northern hybridization to detect the expression level of the target gene, and detect whether the resistant buds or resistant plants are transgenic materials;

S4. Transplantation of transgenic plants: The resistant plants confirmed to contain the target gene are removed from the culture bottles, the root culture medium is washed off, and the plants are transplanted into pots filled with culture medium to obtain successfully transplanted transgenic plants.

Preferably, the disinfection in step S1 is to rinse the young leaves with tap water, soak them in a 0.1% carbendazim aqueous solution for 5-10 minutes, take them out and rinse them with tap water; soak them in a 75% ethanol aqueous solution for 30 seconds, rinse them with sterile water for 3 times, move the young leaves into a 0.1% mercuric chloride aqueous solution containing 0.05% Tween 80 for 12 minutes, rinse them with sterile water for 6 times, and then place the leaves on sterile filter paper to absorb the moisture.

Preferably, the rinsing in step S2 is to rinse the co-cultured explants with a 0.05% by mass Tween 80 sterile aqueous solution for 5 to 6 times, then rinse once with a 500 mg/L cephalosporin sterile aqueous solution, and place the explants on sterile paper to absorb water.

Preferably, in step S2, the explant is immersed in an Agrobacterium tumefaciens solution containing a plant expression vector carrying the target gene for 25 minutes.

Preferably, in the step S2, the resistance bud induction screening medium I is MS medium supplemented with 1 mg/L BA, 0.1 mg/L NAA, 15 mg/L hygromycin, 500 mg/L cephalosporin, 30 g/L sucrose and 8 g/L agar, pH 5.8; the resistance bud induction screening medium II is MS culture supplemented with 1 mg/L BA, 0.1 mg/L NAA, 15 mg/L hygromycin and 250 mg/L cephalosporin, 30 g/L sucrose and 8 g/L agar, pH 5.8.

Preferably, in step S2, the resistant rooting medium is 1/2MS medium supplemented with 25 mg/L hygromycin, 250 mg/L cephalosporin, 30 g/L sucrose and 8 g/L agar, pH 5.8.

Preferably, the culture temperature in steps S1 and S2 is 23-26°C.

Preferably, the Agrobacterium tumefaciens containing the plant expression vector carrying the target gene is constructed by the following method: after inserting the target gene into the expression vector promoter, the constructed plant expression vector carrying the target gene is introduced into the Agrobacterium tumefaciens by the freeze-thaw method, and the Agrobacterium tumefaciens containing the plant expression vector carrying the target gene is obtained through resistance screening.

Preferably, the Agrobacterium tumefaciens bacterial solution containing the plant expression vector carrying the target gene is prepared by the following steps: first suspending the Agrobacterium tumefaciens containing the plant expression vector carrying the target gene in MS medium containing 20 mg/L acetosyringone, the pH of which is 5.2, and culturing until OD600 = 0.4-0.5.

Preferably, the plant expression vector is a pCAMBIA1301 vector (which contains a β-glucuronidase reporter gene gus driven by a CaMV 35S promoter and a hygromycin phosphotransferase screening gene hpt as a screening gene), or a p1390Ubi vector. The p1390Ubi vector is constructed by cutting out the Ubiquitin promoter from the plasmid pAHC27 using HindIII and BamHI and inserting the promoter fragment into the HindIII and BamHI sites of the plasmid pCAMBIA1390, and contains a hygromycin phosphotransferase screening gene hpt driven by a CaMV 35S promoter as a screening gene and a maize Ubiquitin promoter. According to research needs, the target gene can be inserted after the CaMV 35S promoter or the Ubiquitin promoter of the above vectors.

The MS medium is an internationally used medium. Its composition and preparation method can be found in Toshio Murashige, Folke Skoog (1962) A Revised Medium For Rapid Growth And BioAssays With Tobacco Tissue Cultures. Physiollogia Plantarum, 15: 473-497. 1/2MS medium is a medium in which each element in the MS medium is reduced by half.

The invention firstly adopts plant tissue culture technology to obtain a large number of single-seat chicory sterile seedlings for gene transformation; according to the needs of actual research or breeding goals, a plant expression vector containing a target gene driven by the CaMV35S promoter of tobacco cauliflower mosaic virus or the Ubiquitin promoter of corn and a screening marker gene is constructed, the expression vector is introduced into Agrobacterium tumefaciens, the Agrobacterium tumefaciens is co-cultured with leaf blades, petioles and stem explants, and the resistant buds and resistant plants obtained by screening with antibiotics corresponding to the screening marker gene are proved to be transgenic materials by GUS staining and Southern hybridization detection technology. The whole operation process from cutting leaf blades, petioles and stem node explants from sterile seedlings for Agrobacterium tumefaciens infection to obtaining transgenic plants that can be transplanted takes 4 months. By operating 100 leaf explants at a time, about 11 transgenic strains of strains can be obtained on average, each transgenic leaf can produce more than 20 buds after 6 weeks of culture, the rooting rate of the regenerated plants is 100%, and the survival rate of the transgenic plants transplanted into the soil reaches 100%.

The method of the present invention can be used to obtain a large number of new transgenic monosperm germplasms within 4 months, which meets the current urgent needs for monosperm biological research and breeding technology. At the same time, it also provides an effective research method and valuable research materials for the innovation, development, and breeding of excellent new varieties of monosperm germplasm resources, and has significant scientific, ecological, economic and social benefits.

The present invention has the following advantages:

(1) Accelerate scientific research on single-seat moss

Transgenic technology is an important means of plant science research. The transgenic technology provided by the present invention can provide the necessary research technology for scientific research on the gene function, development, reproduction, characteristics, etc. of the monospermum, and promote scientific research on the endangered species of monospermum.

(2) Rapidly create new genetically improved varieties of single-seat chicory

The flower color of the single-seat chicory is peculiar. The method of the present invention can be used to obtain a large amount of new germplasm of the single-seat chicory within 4 months, create a series of mutants, and purposefully improve the traits of the single-seat chicory according to breeding, landscape or ecological needs to develop the ornamental horticultural value of the single-seat chicory.

(3) Efficient single-seat tissue culture plant regeneration system

Previous studies on bud induction of monocotyledonous spatholobi used high concentrations of TDZ, BA, NAA, and IBA, and bud rooting used both NAA and IBA. The monocotyledonous spatholobi tissue culture plant regeneration system established by the present invention is very efficient: 100% of the leaves, petioles, and stem nodes cut from the sterile seedlings cultured by the present invention can be induced to grow buds in the bud induction medium provided by the present invention, and the bud induction medium only uses BA and NAA; the rooting rate of regenerated buds in the rooting medium of the present invention is 100%, and no hormones are added to the rooting medium; 100% of the regenerated plants can be successfully transplanted to soil and survive.

(4) Explant culture for transformation is simple

According to the method provided by the present invention, the sterile seedlings can be sterilely stored under laboratory conditions for a long time, the source of the explants is not restricted, and the genetic modification operation can be carried out at any time.

(5) Strict and effective method for screening transformed plants

The resistant plants obtained by 25 mg/L hygromycin screening were proved to be transgenic materials by GUS staining, Southern hybridization and Northern hybridization experiments. This ensures that the resistant plants obtained by this technical method are transgenic plants and reduces the workload of molecular detection in the later stage.

(6) High transformation efficiency and good repeatability

The technical method provided by the present invention has been repeatedly tested for many times, and the transformation rate has been proved to be 11%-15.2% (Table 4). Since the explants used for transformation are easy to obtain in large quantities, a large number of desired transgenic strains can be obtained in one transformation experiment.

(7) High breeding efficiency

Through the above operations, each positive transformed explant can produce clustered buds, and the clustered buds can be multiplied through subculture. In each subculture cycle (21 days), one leaf can multiply to form more than 20 buds, and all of them can take root and develop into plants. Therefore, a large number of transgenic strains can be obtained within 4 months by applying the method of the present invention, and 100% of the transgenic strains can be successfully transplanted into the soil, ensuring the successful cultivation of varieties cultivated by molecular breeding methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the T-DNA region of a plant expression vector; wherein 35S pro is the promoter of CaMV35S of tobacco cauliflower mosaic virus; and Ubi is the Ubiquitin promoter of corn.

FIG. 2 shows the resistant clusters of buds transformed with pCAMBIA1301 42 days after inoculation on the resistance bud induction screening medium.

FIG. 3 shows the results of transient expression detection of the GUS gene in leaf explants of single-seat Herba Lysimachiae after co-cultivation with Agrobacterium tumefaciens EHA105/pCAMBIA1301 for 3 days.

FIG. 4 shows resistant clustered shoots (42 days; panel A) and resistant shoots (80 days; panel B) transformed with pCAMBIA1301, which were blue stained with GUS.

FIG. 5 shows non-transformed regenerated plants without GUS staining (Panel A) and resistant regenerated plants transformed with pCAMBIA1301 with GUS staining (Panel B).

Figure 6 is the result of Southern hybridization detection of hpt in pCAMBIA1301 transformants; wherein WT is an untransformed strain, showing no hybridization signal; T1, T2, T3, T4, and T5 are pCAMBIA1301 transformants, all of which have hybridization signals and are different transformant strains.

Figure 7 shows the flowering phenotypes of Hd3a plants at 22 days (Figure A), 40 days (Figure B), 67 days (Figure C), and 90 days (Figure D) in the vermiculite matrix during transplantation growth, as well as the wild-type WT regenerated plants and pCAMBIA1301-transfected plants (Figure F) that had not yet flowered at 1 month (Figure E) and 1 year after transplantation.

Figure 8 shows Southern hybridization detection of Hd3a (Figure A) and Northern hybridization detection of Hd3a expression (Figure B); among them, WT is an untransformed strain, showing that no Hd3a-specific hybridization signal and Hd3a expression were detected; H1, H2, H3, and H4 are Hd3a-transformed plants, corresponding to plant samples A, B, C, and D in Figure 7, respectively.

Detailed ways

The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

Example 1

A molecular breeding method for single-seat chicory in this embodiment comprises the following steps:

a. Cultivation of explants for transformation

Take the young leaves of the single-seat chicory plant that grow well, rinse them with tap water, soak them in a 0.1% carbendazim aqueous solution for 5-10 minutes, take them out and rinse them with tap water. Soak them in a 75% ethanol aqueous solution for 30 seconds, rinse them with sterile water three times, move the young leaves into a 0.1% mercuric chloride aqueous solution containing 0.05% Tween 80 and soak them for 12 minutes, rinse them with sterile water six times, and then place the leaves on sterile filter paper to absorb the water. Cut the leaves into 0.6 cm long sections for transformation experiments or inoculate them one by one into bud induction medium, which is MS medium supplemented with 1 mg/L BA, 0.1 mg/L NAA, 30 g/L sucrose and 8 g/L agar, pH 5.8. Culture them under the conditions of temperature 24±1℃, light intensity 50μmol·m ^–2 ·s ^–1 , and light 12h/day. After about 21 days of culture, green callus tissue can be seen, which then develops into buds. When the buds are about 1 cm high, they are cut out and transferred to rooting medium. The rooting medium is 1/2MS medium with 30 g/L sucrose and 8 g/L agar, pH 5.8; the 1/2MS medium refers to the medium obtained by reducing all elements in the MS medium by half. Rooting induction is carried out under the same conditions. After that, 21 days is regarded as one subculture, and buds about 1 cm high are continuously cut off and transferred to rooting medium for culture. Leaves are cut from sterile seedlings about 1.5 cm high as explants for transformation.

b. Preparation of engineered strains containing target gene plant expression vectors

The pCAMBIA1301 vector (the pCAMBIA1301 vector contains the β-glucuronidase reporter gene gus driven by the CaMV 35S promoter and the hygromycin phosphotransferase screening gene hpt as screening genes, and the target gene can be placed behind the CaMV 35S promoter according to the breeding target, FIG1 , and the β-glucuronidase reporter gene gus is used as the experimental test gene in this embodiment) was introduced into Agrobacterium tumefaciens EHA105 by the freeze-thaw method, and then inoculated in a YEP medium containing 50 mg/L kanamycin and 50 mg/L rifampicin, and cultured at 28° C. for 24 hours, and the bacterial solution was collected by centrifugation, and the bacterial cells were suspended in a MS medium (pH 5.2) containing 20 mg/L acetosyringone, OD600=0.4-0.5, thereby obtaining an Agrobacterium tumefaciens EHA105 bacterial solution containing the pCAMBIA1301 vector, which was named Agrobacterium tumefaciens EHA105/pCAMBIA1301.

c. Obtaining regenerated plants containing hygromycin resistance

The leaf explants were immersed in the bacterial solution of Agrobacterium tumefaciens EHA105/pCAMBIA1301 for 25 minutes, then the explants were taken out, placed on sterile paper to absorb the excess bacterial solution, and then the explants were moved to a co-culture medium, wherein the co-culture medium was MS medium supplemented with 1 mg/L BA, 0.1 mg/L NAA, 20 mg/L acetosyringone, 30 g/L sucrose and 8 g/L agar, pH 5.5, and cultured at 25±1°C in the dark for 3 days. The explants after co-cultivation were taken out, rinsed 5-6 times with a 0.05% Tween 80 sterile aqueous solution, and finally rinsed once with a 500 mg/L cephalosporin sterile aqueous solution, placed on sterile paper, dried, and moved to a resistance bud induction screening medium I, which is an MS medium supplemented with 1 mg/L BA, 0.1 mg/L NAA, 15 mg/L hygromycin, 500 mg/L cephalosporin, 30 g/L sucrose and 8 g/L agar, pH 5.8. The explants were placed at 24±1°C and cultured under light for 21 days.

The explants with resistant buds were transferred to resistant bud induction screening medium II, which was MS culture supplemented with 1 mg/L BA, 0.1 mg/L NAA, 15 mg/L hygromycin and 250 mg/L cephalosporin, 30 g/L sucrose and 8 g/L agar, pH 5.8. The medium was placed at 24 ± 1 ° C and cultured under light for 21 days (Figure 2). Fresh resistant bud induction screening medium II was replaced every 21 days, and when the resistant buds were cultured to a height of 1 cm, they were transferred to resistant bud rooting medium, which was 1/2 MS medium supplemented with 25 mg/L hygromycin, 250 mg/L cephalosporin, 30 g/L sucrose and 8 g/L agar, pH 5.8, placed at 24 ± 1 ° C, cultured under light, and the elongation of resistant buds and the production of adventitious roots were observed. Thus, hygromycin-resistant regenerated plants (resistant plants) transformed with pCAMBIA1301 were obtained.

d. Molecular detection of transgenic plants

Since pCAMBIA1301 contains the gus reporter gene, the expression of the gus gene can be detected by staining according to the GUS staining method of Jefferson et al. (1987). The leaf explants, resistant buds, untransformed regenerated plants and transformed resistant plants after co-culture with Agrobacterium tumefaciens EHA105/pCAMBIA1301 for 3 days were immersed in X-Gluc staining solution, kept at 37°C for 3-5 hours, decolorized with 70% ethanol, and the blue coloring was observed. It can be seen that there are obvious blue spots on the surface of the explants (Figure 3) after co-culture with Agrobacterium tumefaciens EHA105/pCAMBI A1301 for 3 days, indicating that Agrobacterium tumefaciens EHA105/pCAMBI A1301 can absorb and infect the explants of the single-seat chicory, and the resistant buds (Figure 4) and resistant plants (Figure 5) also show blue, while the untransformed regenerated plants (Figure 5) do not show blue, indicating that through the above operations, the gus gene has been successfully introduced into the single-seat chicory and stably expressed in the transformed strains. The specific primers for the screening gene hpt were used: HFw (5’-CGATCTTAGCCAGA CGAGCGGGTTC-3’) and HRe (5’-GCTGGGGCG TCGGTTTCCACTATCGG-3’) to synthesize the probe. Through Southern hybridization, hybridization signals were observed in the resistant plants T1, T2, T3, T4, and T5, and the band types were different (Figure 6), indicating that T1, T2, T3, T4, and T5 were all different transgenic strains, while the untransformed plant WT (wild type) had no signal, which once again showed that the hpt gene had been successfully introduced into the genome of the single-seat chicory through the above method.

e. Transplantation of transgenic regenerated plants

The non-transgenic strains and transgenic plants higher than 2 cm were removed from the culture bottles, the root culture medium was washed off, and the plants were planted in a basin filled with vermiculite, covered with plastic wrap, and some small holes were made in the plastic wrap. After 5 days, the survival rate reached 100%. After 31 days of planting, the growth of the two plants was observed and compared. As shown in Fig. 7E and F, the growth of the regenerated plants of the hygromycin-resistant regeneration of pCAMBIA1301 and the untransformed plants was basically the same, indicating that the obtained pCAMBIA1301 did not significantly affect the growth and phenotype of the plants.

Using the method of this embodiment, the entire operation process from cutting leaf explants from sterile seedlings to infecting with Agrobacterium tumefaciens to obtaining transgenic plants that can be transplanted takes 4 months. By operating 100 leaf explants at a time, an average of about 11 transgenic strains can be obtained, and each transgenic leaf can produce more than 20 buds after 6 weeks of cultivation, the rooting rate of the regenerated plants is 100%, and the survival rate of the transgenic plants transplanted into the soil is 100%.

Concentration of hygromycin selection agent in resistance shoot induction medium:

Example 2

The remaining steps of the method of this embodiment are the same as those of Example 1, and the only difference is that the concentration of hygromycin in the resistance bud induction screening medium I and II used in this embodiment is 10 mg/L, and the corresponding screening effect is shown in Table 1.

Example 3

The remaining steps of the method of this embodiment are the same as those of Example 1, and the only difference is that the concentration of hygromycin in the resistance bud induction screening medium I and II used in this embodiment is 20 mg/L, and the corresponding screening effect is shown in Table 1.

Example 4

The remaining steps of the method of this embodiment are the same as those of Example 1, and the only difference is that the concentration of hygromycin in the resistance bud induction screening medium I and II used in this embodiment is 30 mg/L, and the corresponding screening effect is shown in Table 1.

Comparative Example 1

The remaining steps of the method of this comparative example are the same as those of Example 1, with the only difference being that the explants used in this comparative example are stem slices (take plants with a stem width of about 0.5 cm, and cut about 0.1 cm stem slices horizontally as explants), and the corresponding screening results are shown in Table 1.

Comparative Example 2

The remaining steps of the method of this comparative example are the same as those of Example 1, except that the explant used in this comparative example is a petiole (a petiole with a length of about 0.5 cm cut transversely is used as the explant). The corresponding screening results are shown in Table 1.

Parallel cultivation was carried out according to the methods of Example 1, Example 2, Example 3, Example 4, Comparative Example 1 and Comparative Example 2, respectively, with a cultivation time of 42 days. The results are shown in Table 1.

Table 1 Effects of different concentrations of hygromycin on bud induction of leaf explants

Grouping Hygromycin concentration (mg/L) Sample size (number of explants) Bud growth rate after 42 days (%) Example 1 15 75 0 Example 2 10 75 5.3 Example 3 20 75 0 Example 4 30 75 0 Comparative Example 1 15 20 0 Comparative Example 2 15 20 0

As can be seen from Table 1, when the hygromycin dosage was between 15 and 30 mg/L, no buds were produced after 42 days of culture, indicating that 15 mg/L hygromycin can be used in the resistance bud induction screening medium I and II to screen the transformation of leaf, stem and petiole explants.

About Agrobacterium infection time:

Example 5

The remaining steps of the method of this embodiment are the same as those of Example 1, with the only difference being that in step c of this embodiment, the explants are immersed in the bacterial solution of Agrobacterium tumefaciens EHA105/pCAMBIA1301 for 15 minutes, and the corresponding efficiency of obtaining resistant buds is shown in Table 2.

Example 6

The remaining steps of the method of this embodiment are the same as those of embodiment 1, except that in step c, the explants are immersed in the bacterial solution of Agrobacterium tumefaciens EHA105/pCAMBIA1301 for 35 minutes. The corresponding efficiency of obtaining resistant buds is shown in Table 2.

Comparative Example 3

The remaining steps of the method of this comparative example are the same as those of Example 1, with the only difference being that the explants used in this comparative example are stem slices (take plants with a stem width of about 0.5 cm, and cut about 0.1 cm stem slices horizontally as explants). The corresponding efficiency of obtaining resistant buds is shown in Table 2.

Comparative Example 4

The remaining steps of the method of this comparative example are the same as those of Example 1, with the only difference being that the explant used in this comparative example is a petiole (a petiole with a length of about 0.5 cm cut transversely is used as the explant), and the corresponding efficiency of obtaining resistant buds is shown in Table 2.

Comparative Example 5

The remaining steps of the method of this comparative example are the same as those of Example 1, with the only difference being that the explant used in this comparative example is taken from the third leaf from the top of a plant 10 cm high cultured in vermiculite between growth chambers, and the disinfection method is the same as the disinfection method of young leaves in step a of Example 1. The corresponding efficiency of obtaining resistant buds is shown in Table 2.

Parallel cultivation was carried out according to the methods of Example 1, Example 5, Example 6, Comparative Example 3, Comparative Example 4 and Comparative Example 5, respectively. The results are shown in Table 2.

Table 2 Effect of different Agrobacterium infection time on the production rate of resistant buds

As can be seen from Table 2, the infection time of Agrobacterium is 25-35min, the efficiency of GUS transient expression in explants is higher than 70%, the production rate of resistant buds is the highest in Example 1, Example 6 and Comparative Example 3, and the positive rate of GUS in resistant buds is the highest in Example 1. In Comparative Example 5, the explants may be damaged because they are derived from disinfected leaves, which affects the adsorption effect of Agrobacterium. In summary, transgenic buds can be obtained by using leaves, stems, petioles of tissue culture sterile seedlings and leaves of non-tissue culture seedlings as explants for Agrobacterium infection. Among them, the transformation efficiency obtained by using leaves of tissue culture seedlings is the highest, because the stems and petioles of single-seat chicory are extremely short, and the explants collected in the wild must be disinfected, causing damage to the explants, while the leaves of tissue culture seedlings are relatively large. Therefore, using leaves of tissue culture seedlings as transformation explants has the best effect.

Concentration of hygromycin selection agent in rooting medium:

Example 7

The remaining steps of the method of this embodiment are the same as those of Example 1, and the only difference is that the concentration of hygromycin in the resistant bud rooting medium used in this embodiment is 15 mg/L, and the corresponding efficiency of obtaining rooted resistant plants is shown in Table 3.

Example 8

The remaining steps of the method of this embodiment are the same as those of Example 1, and the only difference is that the concentration of hygromycin in the resistant bud rooting medium used in this embodiment is 30 mg/L, and the corresponding efficiency of obtaining rooted resistant plants is shown in Table 3.

Parallel cultivation was performed according to the methods of Example 1, Example 7, and Example 8, respectively. The results are shown in Table 3.

Table 3 Effects of different concentrations of hygromycin on rooting of resistant shoots

As can be seen from Table 3, when the concentration of hygromycin in the rooting medium is 25-30 mg/L, the resistant plants that can grow roots have a GUS positivity rate of 100%. Therefore, the screening dose of hygromycin in the resistance bud rooting medium is 25 mg/L to ensure that the resistant plants obtained are all positive and reduce the workload of molecular detection.

Example 9

The flowering gene Hd3a from rice was introduced into the genome of the single-seat lettuce by the transgenic method of the single-seat lettuce established in Example 1, promoting early flowering of the single-seat lettuce, and creating a new early-flowering germplasm of the single-seat lettuce by using the molecular breeding method of the single-seat lettuce.

The method steps of this embodiment are basically the same as those of Embodiment 1, except that:

a. Preparation of engineered bacteria containing target gene plant expression vector: Preparation of Agrobacterium tumefaciens EHA105/p1390UbiHd3a bacterial solution

Taking the flowering gene Hd3a from rice as the target gene, we constructed a plant expression vector of Hd3a driven by the Ubiquitin promoter from corn by the following method: The Ubiquitin promoter was cut out from the plasmid pAHC27 using HindIII and BamHI, and the fragment was inserted into the HindIII and BamHI sites of the plasmid pCAMBIA1390 to construct the plasmid p1390Ubi. Extract rice leaf mRNA, reverse transcribe into cDNA, design primers Hd3aFw: 5'-AATGAATTC (EcoRI site) ATGGCCGGAAGTGGCAGGGAC-3' and Hd3aRe: 5'-AATGAATTC (EcoRI site) CTAGGGGTAGACC CTCCTG-3' according to the sequence of rice Hd3a gene (GeneBank accession number: Os06g0157700), amplify Hd3a fragment from cDNA, clone the fragment into pGEM-T vector, insert the fragment into EcoRI site of plasmid p1390Ubi after sequencing verification, and obtain the constructed plasmid p1390UbiHd3a after restriction digestion and sequencing verification, the specific structure is shown in Figure 1. The plasmid also contains selection marker gene hpt, which can be used to screen transformed cells. p1390UbiHd3a was introduced into Agrobacterium tumefaciens EHA105 by the freeze-thaw method, and then inoculated into YEP medium containing 50 mg/L kanamycin and 50 mg/L rifampicin, and cultured at 28°C for 24 hours. The bacterial liquid was collected by centrifugation, and the bacterial cells were suspended in MS medium (pH 5.2) containing 20 mg/L acetosyringone, OD600 = 0.4-0.5, thereby obtaining Agrobacterium tumefaciens EHA105 bacterial liquid containing the p1390UbiHd3a vector, which was named Agrobacterium tumefaciens EHA105/p1390Ubi Hd3a.

b. Phenotype of regenerated plants transfected with Hd3a

The Hd3a-transformed plants showed early flowering. Figures 7A-D show the flowering phenotypes of the transgenic plants at 22, 40, 67, and 90 days after being moved to the growth room, respectively. However, the WT untransformed plants and the pCAMBIA1301-transformed plants did not flower after being cultivated in the growth room for one year (Figures 7E-F).

c. Molecular detection of Hd3a transgenic plants

Using the specific primers Hd3aFw: 5'-ATGGCCGGAAGTGGCAGGGAC-3'; Hd3aRe: 5'-CTAGGGGTAGACCCTCCTG-3' of Hd3a to synthesize the probe, the hybridization signal can be observed in the early flowering Hd3a transgenic plants A, B, C, and D by Southern hybridization, and the banding pattern is different (Figure 8A), indicating that A, B, C, and D are all different transgenic lines, while the untransformed plant WT (wild type) has no signal, indicating that the Hd3a gene has been successfully introduced into the genome of the single-seat lettuce by the above method. The results of Northern hybridization (Figure 8B) show that the expression signal of Hd3a can be detected in the Hd3a transgenic plants A, B, C, and D, while the untransformed plant (WT) has no expression of Hd3a. It is proved that the expression of the Hd3a gene promotes the early flowering of the Hd3a transgenic single-seat lettuce plants.

The transformation was carried out three times in parallel according to the methods of Example 1 and Example 9, and the transformation efficiency results are shown in Table 4.

Table 4 Comparison of conversion efficiency of different batches

Grouping batch Sample size (number of leaf explants) PCR amplification hpt positive rate (%) Example 1 1 73 11 Example 1 2 69 14.5 Example 1 3 74 14.9 Example 9 1 66 15.2 Example 9 2 82 12.2 Example 9 3 79 11.4

It can be seen from Table 4 that the single-seat Morinda citrifolia transgenic method established by the present invention has good repeatability and the transformation frequency exceeds 11%.

Claims (7)

1. A molecular breeding method for single-seat chicory, characterized in that it comprises the following steps: S1. Cultivation of explants for transformation: the explants are leaves of regenerated plants of the monocotyledonous plant; the regenerated plants are prepared by the following steps: sterilizing young leaves of the monocotyledonous plant, cutting them into 0.5-0.8 cm long segments, inoculating them into bud induction medium, culturing them until green callus appears and then develops into buds, and when the buds are up to 1 cm high, cutting them out and transferring them to rooting medium for culturing, inducing rooting to obtain regenerated plants; The bud induction medium is MS medium supplemented with 1 mg/L BA, 0.1 mg/L NAA, 30 g/L sucrose and 8 g/L agar, pH 5.8; The rooting medium is 1/2MS medium supplemented with 30 g/L sucrose and 8 g/L agar, pH 5.8; The disinfection comprises the following steps: rinsing the young leaves with tap water, soaking them in a 0.1% carbendazim aqueous solution for 5-10 minutes, taking them out and rinsing them with tap water; soaking them in a 75% ethanol aqueous solution for 30 seconds, and then rinsing them with sterile water for 3 times; then transferring the young leaves into a 0.1% mercuric chloride aqueous solution containing 0.05% Tween 80 for 12 minutes, and then rinsing them with sterile water for 6 times; and then placing the leaves on sterile filter paper to absorb the water; S2. Transformation to obtain resistant plants: soak the explants prepared in step S1 in a culture medium of Agrobacterium tumefaciens containing a plant expression vector carrying the target gene for 25 minutes, then take out the explants, absorb the excess culture medium and transfer the explants to a co-culture medium, culture them in the dark for 3 days, then take out the co-cultured explants, rinse them and transfer them to a resistance bud induction screening medium I, culture them under light, and after the resistance buds grow, transfer the resistance buds to a fresh resistance bud induction medium II, and when the resistance buds are up to 1 cm, transfer them to a resistance bud rooting medium, culture them under light, and allow the resistance buds to elongate and produce adventitious roots, thereby obtaining resistant plants; The co-culture medium is MS medium supplemented with 1 mg/L BA, 0.1 mg/L NAA, 20 mg/L acetosyringone, 30 g/L sucrose and 8 g/L agar, pH 5.5; The resistant bud induction screening medium I is MS medium supplemented with 1 mg/L BA, 0.1 mg/L NAA, 15 mg/L hygromycin, 500 mg/L cephalosporin, 30 g/L sucrose and 8 g/L agar, pH 5.8; The resistance bud induction screening medium II is MS medium supplemented with 1 mg/L BA, 0.1 mg/L NAA, 15 mg/L hygromycin and 250 mg/L cephalosporin, 30 g/L sucrose and 8 g/L agar, pH 5.8; The resistance rooting medium is 1/2MS medium supplemented with 25 mg/L hygromycin, 250 mg/L cephalosporin, 30 g/L sucrose and 8 g/L agar, pH 5.8; The rinsing is to rinse the co-cultured explants with a 0.05% Tween 80 sterile aqueous solution for 5 to 6 times, then rinse once with a 500 mg/L cephalosporin sterile aqueous solution, and place the explants on sterile paper to absorb water; S3. Molecular detection of resistant plants: Use the selection marker gene, reporter gene or target gene sequence on the plant expression vector to perform Southern hybridization to detect whether the gene has been transferred into the genome of the single-seat lettuce, use GUS staining or Northern hybridization to detect the expression level of the target gene, and detect whether the resistant buds or resistant plants are transgenic materials; S4. Transplantation of transgenic plants: The resistant plants confirmed to contain the target gene are removed from the culture bottles, the root culture medium is washed off, and the plants are transplanted into pots filled with culture medium to obtain successfully transplanted transgenic plants. 2. The method according to claim 1, characterized in that the culture temperature in steps S1 and S2 is 23-26°C. 3. The method according to claim 1, characterized in that the Agrobacterium tumefaciens containing the plant expression vector carrying the target gene is constructed by the following method: after the target gene is inserted into the promoter of the expression vector, the constructed plant expression vector carrying the target gene is introduced into the Agrobacterium tumefaciens by the freeze-thaw method, and the Agrobacterium tumefaciens containing the plant expression vector carrying the target gene is obtained through resistance screening. 4. The method according to claim 1 is characterized in that the Agrobacterium tumefaciens bacterial solution containing the plant expression vector carrying the target gene is prepared by the following steps: first suspending the Agrobacterium tumefaciens containing the plant expression vector carrying the target gene in MS medium containing 20 mg/L acetosyringone, whose pH is 5.2, and culturing until OD600 = 0.4-0.5. 5. The method according to claim 1, characterized in that the plant expression vector is pCAMBIA1301, pCAMBIA1390 or p1390Ubi vector. 6. Use of the molecular breeding method for single-seat lettuce according to any one of claims 1 to 5 in obtaining a new variety of single-seat lettuce that blooms early through genetic modification. 7. The use according to claim 6, characterized in that the rice Hd3a gene is transferred into the genome of the single-seat chicory as a target gene.