CN117322323A - Method for improving regeneration and conversion efficiency of sweet corn - Google Patents

Abstract

The invention discloses a method for improving the regeneration and conversion efficiency of sweet corn. Firstly, through preliminary screening of the regeneration capacity of different sweet corn varieties, the genotype of the sweet corn variety Xuesu 7401 is determined to be suitable for genetic transformation, but the regeneration capacity of the variety is weaker. Then, the sweet corn variety snow sweet 7401 is hybridized with HiII, and a series of processes such as backcrossing and selfing are performed to cultivate a sweet corn genotype material with strong regeneration capacity and high genetic transformation efficiency, and the applicant names the genotype material as TH44. Compared with the original parent snow sweet 7401, the genotype material TH44 has the advantage that the callus regeneration and transformation efficiency are improved by 2 times. Using TH44 as a transforming receptor, vectors with GFP have been introduced into this receptor material and a partial transgenic T has been obtained ₀ And (5) replacing plants.

Description

A method for improving sweet corn regeneration and transformation efficiency

Technical field

The invention relates to the technical field of plant genetic engineering, and in particular to a method for improving the regeneration and transformation efficiency of sweet corn.

Background technique

Sweet corn is deeply loved by consumers because of its rich nutrition and good taste. As a new type of fruit and vegetable crop, it plays an important role in improving residents' dietary structure and earning foreign exchange from exports. Zhejiang Province is a major sweet corn planting and consumption province, and the demand for high-quality sweet corn varieties is very high. However, factors such as narrow germplasm resources and lagging breeding technology severely restrict the high-quality development of the sweet corn industry in our province.

As an emerging biotechnology, genetically modified technology provides a new technical solution for improving crop yield, quality and resistance. Although many corn genetic transformation methods have been disclosed in the prior art, different genotypes are still important factors limiting the efficiency of corn genetic transformation, and sweet corn genetic transformation is also limited by genotypes. Currently, the receptors suitable for genetic transformation of sweet corn are very limited and the efficiency is very low.

Contents of the invention

In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to overcome the shortcomings of poor regeneration ability, low transformation efficiency, and long transformation cycle in existing genetic transformation of sweet corn, and provide a method for sweet corn regeneration and transformation efficiency.

In order to achieve the above-mentioned object of the invention, the technical solutions adopted by the present invention are:

A method for improving sweet corn regeneration and transformation efficiency is provided, which includes the following steps:

A sweet corn genotype material with strong regeneration ability and high genetic transformation efficiency was developed by crossing the sweet corn variety Xuetian 7401 with HiII, followed by backcrossing and continuous selfing, and then used it for transformation.

Further, it specifically includes the following steps:

A. Use the induction medium to test the regeneration ability of young embryos of different sweet corn varieties, including the callus induction efficiency, callus proliferation rate, callus growth status, etc., to obtain test materials with strong regeneration ability, and use the obtained The materials were selected for subsequent differentiation experiments.

B. Use the callus differentiation medium to test the callus of different sweet corn varieties screened in the early stage, including the callus proliferation rate, callus differentiation time and efficiency, etc., and obtain test materials with strong regeneration ability to be selected for subsequent cultivation. test.

C. Cross the selected sweet corn variety Xuetian 7401 with HiII, and the F1 generation obtained will be backcrossed to 7401 twice, and the BC2F1 offspring will be self-crossed for 2 generations to select sweet corn self-crosses with strong regeneration ability. Line TH44 was used for transformation.

Further, Agrobacterium-mediated genetic transformation method is used to transform TH44. The specific method includes the following sub-steps:

(1) Select corn cobs with normal development and full kernels 9-12 days after pollination, remove the young embryos after surface disinfection, and immerse the young embryos in the infection medium containing Agrobacterium;

(2) Move the infected immature embryos to a co-culture medium and culture them in the dark;

(3) Move the young embryos to the selection medium without bialaphos and continue culturing for about 2-3 weeks;

(4) Move the differentiated immature embryos to a selection medium supplemented with bialaphos and continue culturing until new calli grow;

(5) Move the new callus to the differentiation medium and differentiate to form small buds;

(6) Move the regenerated seedlings to the rooting medium;

(7) When the seedlings reach the bottle cap, you can open the seedlings and transplant them to obtain genetically modified corn.

Further, the various culture medium components and proportions in the above-mentioned culture medium are as follows:

Infection medium: 1/2MS+sucrose 68.5g/L+glucose 36g/L+L-proline 0.115g/L+acetosyringone 200mM;

Co-culture medium: 1/2MS + sucrose 20g/L + glucose 10g/L + proline 0.115g/L + thiamine hydrochloride 0.5mg/L + AgNO3 20mM + 2,4-D 0.5mg/L + picloram 2.2mg/L + acetyl Syringone 200mM;

Induction medium: MS+sucrose 30g/L+proline 2g/L+thionine hydrochloride 0.5mg/L+AgNO320Mm+hydrolyzed casein 0.3g/L+2,4-D 0.5mg/L+Toxidine 2.2mg/L+Special Medin 200mg/L;

Screening medium: MS + sucrose 30g/L + proline 2g/L + thiamine hydrochloride 0.5mg/L + AgNO320mM + hydrolyzed casein 0.3g/L + 2,4-D 0.5mg/L + picloram 2.2mg/L + Temei Stingin 200mg/L + bialaphos 0.5-50mg/L;

Differentiation medium: MS+sucrose 20g/L+6-BA 3.5mg/L+copper sulfate 10mM+MES 0.5g/L+timentin 200mg/L;

Rooting medium: MS+sucrose 20g/L+MES 0.5g/L+IBA 0.2mg/L.

The beneficial effects of the present invention are:

The transgenic sweet corn inbred line TH44 obtained by the present invention has the advantages of short plants, early flowering, and short growth period. After being used as a sweet corn transgenic receptor, the transgenic material obtained can be directly used as an inbred line for sweet corn breeding, which can greatly shorten the breeding period. Sweet corn breeding cycle. In addition, the transformation efficiency and regeneration capacity of TH44 are significantly higher than other sweet corn varieties, which is of great significance for the further application and promotion of transgenic technology in sweet corn. The present invention selects sweet corn inbred lines with high regeneration and differentiation capabilities through the mode of screening-crossing-backcrossing-selfing purification, which also has certain significance in guiding the genetic transformation research of other plants.

Description of drawings

Figure 1 is a flow chart of the breeding process of the receptor material TH44 of the present invention;

Figure 2 is a map of the plasmid vector pRF-GFP constructed in the present invention;

Figure 3 is a schematic diagram of the regenerative callus of F1 and its progeny TH2 and TH44 during the breeding process of the present invention and the positive callus obtained using TH44 as a receptor.

Detailed ways

The following examples define the present invention, describe how to identify the young embryo induction and differentiation capabilities of sweet corn, and provide an application example of TH44 as an excellent transgene receptor. From the following description and these examples, those skilled in the art can determine the basic characteristics of the present invention, and without departing from the spirit and scope of the present invention, can make various changes and modifications to the present invention to make it suitable for different applications. purposes and conditions.

Example 1 Preliminary evaluation test of regeneration ability of different sweet corn immature embryos on induction medium

In order to verify the technical route of the present invention, three sweet corn varieties (Xuetian 7401, purchased from Fujian Jinmiao Seed Industry, Zhetian 20, a gift from the Breeding Research Office of the Corn and Characteristic Dry Grain Research Institute, Zhejiang Academy of Agricultural Sciences, Zhejiang Taitian 928, a gift from the Cultivation Research Office of the Maize and Specialty Dry Grain Research Institute, Zhejiang Academy of Agricultural Sciences) and 6 sweet corn inbred lines (T21, germplasm preserved in the laboratory, T11, germplasm preserved in the laboratory, T12, germplasm preserved in the laboratory , T16, germplasm preserved in the laboratory, T39, germplasm preserved in the laboratory) were compared for regeneration ability. The induction and differentiation abilities of sweet corn immature embryos were compared as shown in Table 1.

The steps are as follows:

Cultivation of young embryos: Pollination is carried out after corn blooms. Take the female ears about 9-12 days after pollination, peel off the corn bracts, surface sterilize with 75% alcohol, and then sterilize the cobs twice in 5%-10% sodium hypochlorite. , 15 minutes each time, rinse 5 times with sterilized water after taking out and set aside.

Establishment of induction culture system for sweet corn immature embryo callus: Peel the immature embryos from the fruit kernels with a knife and place them in liquid induction medium for later use. After all immature embryos are peeled off, replace the liquid for induction culture. base, place it on sterilized filter paper, absorb the excess liquid, and blow dry for 10-30 minutes until there is no water stain on the surface. Then move the young embryos to the corn induction medium, culture them in the dark at 26°C for 14 days, and observe the healing Injury-inducing ability.

Establishment of the differentiation culture system of sweet corn callus: After the induced callus is subcultured for 2-3 weeks, it is placed on the corn differentiation medium for differentiation. During this period, each generation is about 2-3 weeks, and the growth is selected. The vigorous young shoots will grow to about 3-4cm in height, be transferred to the rooting medium, and placed under light at 28°C for about 2-3 weeks.

Research results show that in key indicators of callus induction and differentiation ability, Xuetian 7401 is better than the province's independently cultivated varieties (ZT20, ZTT928) and sweet corn inbred lines (see Table 1).

Table 1 Comparison of regeneration abilities of different sweet corns

Example 2: TH44 breeding process and comparison of transformation and regeneration efficiency of different strains of the same generation

After crossing HiII (a gift from Zhejiang University) and 7401, the sweet corn inbred line population was purified and isolated as the recipient. Plants with excellent agronomic properties were selected for pollination. The cobs 9-12 days after pollination were taken for callus induction and differentiation. , the method is the same as in Example 1.

Research results show that TH44 has better callus induction and differentiation capabilities than other sweet corn inbred lines (see Figure 3).

Example 3: Agrobacterium-mediated GFP genetic transformation using TH44 as the transformation receptor

Agrobacterium strain EHA105 and plasmid vector are used for genetic transformation of sweet corn. The T-DNA structure of the plasmid is shown in Figure 2. The procedure for Agrobacterium transformation of sweet corn inbred line TH44 is as follows: take the cobs 9-12 days after pollination, remove the seed coat and part of the endosperm after disinfection, place the resulting young embryos in liquid induction medium, and wait until all are taken out. , replace with a new liquid induction medium,

Inoculate the Agrobacterium bacteria liquid suspended in co-culture medium with OD600=0.5. After infection for 10 minutes, use sterile filter paper to absorb the bacterial liquid on the surface of the young embryos, and place the young embryos in a culture medium containing co-culture media. Dish, co-culture for 3 days at 25°C, put the young embryos into sterile water containing cephalosporin (250mg/L), rinse 3-5 times, absorb the water stains on the surface, and connect it to the screening medium, every 2-3 Succession is carried out once every week until resistance and healing are obtained. The resistant callus differentiated from sweet corn was used to detect the transient expression of GFP gene using stereofluorescence microscopy. The stereofluorescence microscope model is Nikon L300, the filter is GFP, and the excitation light source is blue light with a wavelength of 480 nm (Figure 3).

The present invention preliminarily screens the regeneration abilities of different sweet corn varieties, and then re-screens the transformation abilities of different sweet corn varieties through tissue culture and genetic transformation experiments, and determines that the genotype of the sweet corn variety Xuetian 7401 is suitable for genetic transformation, but The regeneration ability of this variety is relatively weak. The applicant used the cross between the sweet corn variety Xuetian 7401 and HiII, followed by a series of processes such as backcrossing and continuous selfing to cultivate a sweet corn genotype material with strong regeneration ability and high genetic transformation efficiency. The sweet corn genotype is a A good genetically modified material, the applicant named this genotype material TH44. Compared with its original parent Xuetian 7401, the genotype material TH44 has twice the callus regeneration and transformation efficiency. Using TH44 as the transformation receptor, the vector carrying GFP has been introduced into this receptor material, and some transgenic T0 generation plants were obtained.

It is obvious to those skilled in the art that the present invention is not limited to the details of the above-described exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be regarded as illustrative and non-restrictive from any point of view, and the scope of the present invention is defined by the appended claims rather than the above description, and it is therefore intended that all claims falling within the claims All changes within the meaning and scope of equivalent elements are included in the present invention.

In addition, it should be understood that although this specification is described in terms of implementations, not each implementation only contains an independent technical solution. This description of the specification is only for the sake of clarity, and those skilled in the art should take the specification as a whole. , the technical solutions in each embodiment can also be appropriately combined to form other implementations that can be understood by those skilled in the art.

Claims (3)

1. A method for improving the regeneration and conversion efficiency of sweet corn, which is characterized by comprising the following steps:

the sweet corn variety Xuesweet 7401 is hybridized with HiII, then backcrossed and continuously selfed to cultivate the sweet corn genotype material with strong regeneration capacity and high genetic transformation efficiency, and the sweet corn genotype material is used for transformation.

2. The method for improving the regeneration and transformation efficiency of sweet corn according to claim 1, which comprises the following steps:

A. testing the regeneration capacity of young embryos of different sweet corn varieties by using an induction culture medium to obtain a test material with strong regeneration capacity, and selecting the obtained material into a subsequent differentiation test;

B. the callus differentiation culture medium is utilized to test the callus of different sweet corn varieties screened in the earlier stage, and the test material with strong regeneration capacity is obtained to be selected for the subsequent cultivation test;

C. the selected sweet corn variety Xuesweet 7401 is hybridized with HiII, the F1 generation is obtained, the obtained product is backcrossed with 7401 for 2 times, the BC2F1 generation is subjected to selfing for 2 generations, and the sweet corn inbred line TH44 with strong regeneration capacity is selected for transformation.

3. The method for improving the regeneration and transformation efficiency of sweet corn according to claim 2, wherein the agrobacterium-mediated genetic transformation method is used for transforming TH44, and the specific method comprises the following sub-steps:

(1) Selecting maize cobs with normal development and full seeds after pollination for 9-12 days, taking young embryos after surface sterilization, and invading the young embryos into agrobacterium tumefaciens bacteria liquid for infection;

(2) Transferring the infected young embryo to a co-culture medium for dark culture;

(3) Transferring the young embryo to a rest culture medium for continuous culture for about 2-3 weeks;

(4) Transferring the differentiated young embryo to a screening culture medium added with bialaphos for continuous culture until a new callus grows;

(5) Transferring the new callus to a differentiation medium, and differentiating to form buds;

(6) Transferring the regenerated plantlets to a rooting culture medium;

(7) And (5) lifting the seedlings to a bottle cap, opening the seedling, and transplanting to obtain the transgenic corn.