Method for obtaining transgenic wheat by impregnating young ears through agrobacterium tumefaciens

A technique of impregnating transgenic wheat and Agrobacterium, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problems of unsuitable genetic transformation of leading winter wheat varieties, unsatisfactory transformation efficiency of transgenic wheat, etc., and save manpower The effect of material resources, avoiding a long cycle, and simple and convenient operation

Inactive Publication Date: 2013-10-09
HEBEI AGRICULTURAL UNIV.
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Problems solved by technology

However, Ye Xingguo et al. (2011) referred to the method of Zale et al. (2009) to carry out Agrobacterium infection and transformation on many current leading winter wheat varieties such as 987 and Kenong 199 in the northern winter wheat region of my country, but no transformed grains or plants were obtained, indicating that The method of Zale et al. (2009) may not be suitable for the genetic transformation of the current leading winter wheat varieties in my country, and technical improvement needs to be carried out according to the characteristics of my country's wheat
Moreover, the transformation efficiency of transgenic wheat obtained by the Agrobacterium-mediated young ear method is not ideal, and the varieties used are mainly spring wheat.

Method used

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  • Method for obtaining transgenic wheat by impregnating young ears through agrobacterium tumefaciens
  • Method for obtaining transgenic wheat by impregnating young ears through agrobacterium tumefaciens
  • Method for obtaining transgenic wheat by impregnating young ears through agrobacterium tumefaciens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Infection of young ears with Agrobacterium to obtain transgenic wheat with OsDREB2.2 transcription factor

[0027] 1. Preparation of Plant Material

[0028] The wheat varieties Jimai 38 and Zhoumai 16 were planted in the laboratory or in the field, and managed until heading began.

[0029] 2. Transformation of Agrobacterium

[0030] 1) Single clones of Agrobacterium tumefaciens EHA105 and GV3101 were respectively picked and placed in 10 ml of LB liquid medium (containing the corresponding antibiotic: Rif 30 mg / ml), and cultured overnight at 28° C. with shaking.

[0031] 2) Take 2ml of the overnight culture and inoculate it into 50ml LB (containing Rif30mg / ml) liquid medium, shake and culture at 28°C until OD 600 0.6-0.7.

[0032] 3) Divide the bacterial solution into four sterilized 10ml tubes and place in ice bath for 10min. Then at 4°C, 5000rpm, centrifuge for 5min. Remove supernatant and pellet with 200 μl 20 mM ice-cold CaCl 2 Resuspend, centrifuge a...

Embodiment 2

[0046] Example 2 Study on the Transformation Efficiency of Transfection OsDREB2.2 Transcription Factor Gene Wheat by Infecting Young Ears with Agrobacterium

[0047] Target gene identification was carried out on the plant obtained in Example 1 and its second-generation seeds, and the transformation and inheritance of the target gene were studied. The results are shown in Table 1. It can be seen that the transformation rate of Zhoumai 16 is 92.3% according to the number of transformed plants, and that of Jimai 38 is 65.5%. According to the number of transformed seeds, the positive rate of the target gene is 82.3% for Zhoumai 16 and 28.3% for Jimai 38. %. T for each transgenic plant 1 Positive plants ( Figure 5 ), indicating that the transformed target gene was stably inherited. The transformation efficiency of the transgenic wheat obtained by the invention is greatly improved, and relatively satisfactory genetic stability is obtained.

Embodiment 3

[0048] Example 3 Obtaining OsDREB2.2 Transcription Factor Gene Wheat by Infecting Young Ears at Different Stages with Agrobacterium

[0049] Using the method described in Example 1, Agrobacterium infection was carried out at different stages of the wheat ear heading stage, and the results are shown in Table 2. It can be seen from Table 2 that if the dipping is carried out before the young ears of wheat are sheathed, the wheat develops abnormally and cannot set seeds; when the dipping is carried out when the young ears are partly sheathed, the wheat develops normally, sets seeds, and transgenic plants can be obtained.

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Abstract

The invention relates to the field of plant transgenic engineering, in particular to a method for obtaining transgenic wheat by impregnating young ears through agrobacterium tumefaciens. The method for obtaining the transgenic wheat by impregnating the young ears through the agrobacterium tumefaciens comprises the following steps of: 1) culturing agrobacterium tumefaciens strains containing expression vectors which carry target genes, and suspending the strains by using an impregnating culture medium; and 2) selecting the wheat at a heading stage for transformation, removing underdeveloped small ears at the top ends and the tail ends of the young ears, shearing about 1 / 3 of the top ends of the wheat, and soaking the young ears of the wheat into the impregnating culture medium containing the agrobacterium tumefaciens. The method is easy and convenient to operate, saves manpower and material resources, and is high in transformation rate which can reach 26.3 to 82.3 percent; and the impregnated plants can normally grow and fruit, so the method has significance for theoretical research and genetic breeding practice on the aspect of wheat gene engineering.

Description

technical field [0001] The invention relates to the field of plant transgenic engineering, in particular to a method for obtaining transgenic wheat by infecting young ears with Agrobacterium. Background technique [0002] Wheat is an annual or perennial herbaceous plant of the genus Tritucum in the Gramineae family. It is an important food crop that is widely planted in the world and its total output is second only to corn. At present, the breeding of new wheat varieties still mainly depends on conventional breeding. Due to the long cycle of conventional hybrid breeding, single means, difficulty in interspecific hybridization, low efficiency and poor predictability, the genetic background of hybrid offspring is narrow and the difficulty of breeding is increased, making it difficult to improve the level of wheat breeding and the progress of breeding is slow. [0003] Utilizing modern genetic engineering technology to improve wheat varieties is a new direction of current whea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 郎明林王维杨学举刘桂茹温树敏张贵友
Owner HEBEI AGRICULTURAL UNIV.
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