Agrobacterium tumefaciens-mediated sugarcane callus efficient genetic transformation method

A genetic transformation method, Agrobacterium-mediated technology, applied in the field of high-efficiency genetic transformation of sugarcane callus mediated by Agrobacterium, can solve the problems of difficulty in improving the overall transformation efficiency, plant callus damage and death, increased workload, etc., to achieve improved The effects of survival rate and differentiation ability, improvement of bacterial transformation activity, and improvement of accuracy

Active Publication Date: 2022-07-22
SUGARCANE RES INST OF YUNNAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, one-sided, single-item changes are difficult to improve the overall conversion efficiency, but are likely to be counterproductive
For example, if the infiltration level of Agrobacterium is too high, it will cause the excessive growth of Agrobacterium in the subsequent process and the death of plant callus; if the selection pressure of antibiotics or herbicides is increased by overemphasizing the selection of resistant callus, it will inhibit callus differentiation; Injury differentiation reduces the selection pressure, which will greatly increase the workload, and it is easy to generate chimeras

Method used

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  • Agrobacterium tumefaciens-mediated sugarcane callus efficient genetic transformation method
  • Agrobacterium tumefaciens-mediated sugarcane callus efficient genetic transformation method
  • Agrobacterium tumefaciens-mediated sugarcane callus efficient genetic transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Step 1, sugarcane callus induction:

[0043] Take sugarcane (respectively use sugarcane cultivar (Saccharum spp.hybrid) ROC22 and Yuncane 05-51 two varieties) 1-5cm section (preferably 3cm) above the top growth point, peel off the outer mature leaves layer by layer, and work in ultra-clean The surface of Taichung was disinfected by spraying alcohol, and the inner diameter of 1cm young leaf rolls was collected, cut into 1-2mm (preferably 1mm) thick leaf discs, and inoculated into callus induction medium. The cells were cultured in the dark at 28°C, and subcultured once every 2-3 weeks (preferably 2 weeks). Those with dry surface, firm texture and white to light yellow color were used for genetic transformation.

[0044] The above-mentioned callus induction medium was MS+2,4-D 3.0mg / L+sucrose 30g / L+agar powder 8.0g / L, pH 5.8, sterilized by high pressure moist heat at 121°C.

[0045] Step 2, construction of EGFP-Bar double marker screening system and transformation of en...

Embodiment 2

[0068] Step 1 is the same as Step 1 of Embodiment 1.

[0069] Step 2 is the same as Step 2 of Embodiment 1.

[0070] The difference between step 3 and step 3 of Example 1 is that the dip-dyeing medium is 1 / 2MS+2,4-D 3.5mg / L+sucrose 20g / L+glucose 10g / L+AS 40mg / L, pH 5.2.

[0071] Step 4, sugarcane callus intensified dip:

[0072] The sugarcane callus was cut to a size of 5mm, the surface was dried, and then transferred into the infusion solution for intensive infusion. Tween-20 with a volume ratio of 0.1% was added, incubated at 28° C. and 100 rpm in the dark for 15 min, followed by 40 kHz ultrasonic water bath for 10 min at room temperature, 44 kPa vacuum treatment for 5 min, and dark for 5 min.

[0073] The difference between step 5 and step 5 of Example 1 is: CCM medium is 1 / 2MS+2,4-D 2.0mg / L+citric acid 300mg / L+sucrose 20g / L+glucose 10g / L+AS 20mg / L, pH 5.2.

[0074] The difference between step 6 and step 6 of embodiment 1 is:

[0075] CSM medium is MS+2,4-D 2.0mg / L+citri...

Embodiment 3

[0079] Step 1 is the same as Step 1 of Embodiment 1.

[0080] Step 2 is the same as Step 2 of Embodiment 1.

[0081] The difference between step 3 and step 3 of Example 1 is that the dip-dyeing medium is 1 / 2MS+2,4-D 3.0mg / L+sucrose 15g / L+glucose 15g / L+AS 40mg / L, pH 5.2.

[0082] Step 4, sugarcane callus intensified dip:

[0083] The sugarcane callus was cut to a size of 4 mm, the surface was dried, and then transferred into the infusion solution for intensive infusion. Tween-20 with a volume ratio of 0.1% was added, incubated at 28° C. and 100 rpm in the dark for 10 min, followed by a 40 kHz ultrasonic water bath for 5 min at room temperature, 32 kPa vacuum treatment for 10 min, and a dark stand for 10 min.

[0084] The difference between step 5 and step 5 of Example 1 is: CCM medium is 1 / 2MS+2,4-D 2.0mg / L+citric acid 250mg / L+sucrose 15g / L+glucose 15g / L+AS 20mg / L, pH 5.2.

[0085] The difference between step 6 and step 6 of embodiment 1 is:

[0086] CSM medium is MS+2,4-D...

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Abstract

The invention discloses an efficient genetic transformation method for agrobacterium-mediated sugarcane calluses, and belongs to the field of tissue culture. According to the invention, by enhancing the Agrobacterium tumefaciens Vir gene induction activation level and the bacterial liquid dip dyeing strength of sugarcane calluses, the occurrence frequency of transgenic events is greatly improved; an antioxidant is added into subsequent co-culture, screening and differential culture media to inhibit callus browning caused by agrobacterium-enhanced dip dyeing, so that the survival rate and the differentiation capacity of the dip-dyed calluses are obviously improved; a double-marker screening system with a green fluorescent protein marker EGFP gene and a herbicide glufosinate-ammonium resistance marker Bar gene at the same time is adopted, positive transgenic calluses are visually and nondestructively screened by using an EGFP marker, positive transgenic differentiated seedlings are screened by using Bar marker resistance, the accuracy of transgenic event screening is remarkably improved, and the screening efficiency is improved. And finally, the efficient genetic transformation of the agrobacterium tumefaciens mediated sugarcane callus is realized.

Description

technical field [0001] The invention relates to the field of tissue culture, in particular to a method for efficient genetic transformation of sugarcane callus mediated by Agrobacterium. Background technique [0002] Sugarcane is the most important sugar crop and an important source of bioethanol in my country and the world. The continuous genetic improvement of sugarcane varieties is the foundation and key to the healthy development of the industry. However, sugarcane is a highly heterozygous allopolyploid and aneuploid vegetatively propagated crop with complex genetic background and huge genome, making conventional cross-breeding very difficult. At the same time, problems such as difficulty in inducing flowering of sugarcane parents and lack of flowering period further limit the efficiency of trait improvement in cross-breeding. In contrast, genetic engineering breeding can achieve targeted genetic improvement of plant traits without causing large-scale genetic recombina...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/65A01H5/00A01H6/46A01H4/00
CPCC12N15/8205C12N15/65A01H4/008
Inventor 李纯佳刘新龙李旭娟吴转娣胡鑫田春艳
Owner SUGARCANE RES INST OF YUNNAN ACADEMY OF AGRI SCI
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