Method for establishing carnation agrobacterium-mediated efficient transfection system

Abstract

The invention discloses a method for establishing a carnation agrobacterium-mediated efficient transfection system. The method comprises: step (1) a seed germination aseptic seedling stage; step (2) an aseptic seedling propagation stage; step (3) an agrobacterium culture stage; step (4) an invasion and co-culture stage; step (5) a screening stage; step (6) a rooting stage; and step (7) a positivedetection stage. A carnation agrobacterium-mediated efficient transfection system is smoothly established and makes full preparation for cultivating a new carnation variety through transgenic technology, so that a new variety that cannot be cultivated through a traditional breeding manner is obtained. The method is high in frequency of callus induction and good in preservation effect, provides anexcellent receptor for genetic transformation, and is high in conversion efficiency and suitable for massive industrial production and business breeding.

Description

technical field

[0001] The invention belongs to the technical field of plant transformation mediated by Agrobacterium, in particular to a method for establishing a high-efficiency transfection system mediated by Agrobacterium carnation. Background technique

[0002] Carnation (Dianthus caryophyllus) is a model of beauty and elegance with exquisite body, colorful and elegant, dignified and generous, and fragrant and quiet. People often use carnations as gifts for their mothers, and use them to express their love and blessings to their mothers. With the rise of Mother's Day, carnations have become the most popular flower in the world. Carnation is the main export species of Kenya and the largest export flower species of Colombia in the Americas. It is widely cultivated in Japan, South Korea, Malaysia and other countries in Asia. In Europe, countries such as Germany, Hungary, Italy, Poland, Spain, Turkey, the United Kingdom and the Netherlands also have a large scale of culti...

Examples

Embodiment 1

[0051] Example 1: Callus induction and Agrobacterium-mediated high-efficiency transfection of "Xuewu" carnation

[0052] Step 1 Seed germination aseptic seedling stage: Rinse the seeds in sterile water for 3 times, pour out the sterile water, add 75% ethanol, shake for 30 seconds, pour out the ethanol, wash with sterile water for 3 times, pour out the sterile water , add 0.1% mercuric chloride, soak for 5 minutes, pour out the mercuric chloride and recycle, wash the seeds 8 times with sterile water, put the seeds on the filter paper and blow dry. Expose half of the seeds to the air, and half-embedded inoculated on the base medium SH+agar 8g L -1 + sucrose 20g·L -1 , pH 5.8, autoclaved at 121°C for 20min. Light culture at 22-24°C, light for 12 hours a day, light intensity 1800-2000lx;

[0053] SH basic formula is: Potassium nitrate 2.5g L -1 , magnesium sulfate 0.195g L -1 , ammonium dihydrogen phosphate 0.3g·L -1 , calcium chloride 151mg·L -1 , Glycine 2mg·L -1 , inosi...

Embodiment 2

[0076] Example 2: Induction of callus and Agrobacterium-mediated high-efficiency transfection of "Snow White" carnation

[0077] Step 1 Seed germination aseptic seedling stage: Rinse the seeds in sterile water for 3 times, pour out the sterile water, add 75% ethanol, shake for 30 seconds, pour out the ethanol, wash with sterile water for 3 times, pour out the sterile water , add 0.1% mercuric chloride, soak for 5 minutes, pour out the mercuric chloride and recycle, wash the seeds 8 times with sterile water, put the seeds on the filter paper and blow dry. Expose half of the seeds to the air, and inoculate them half-embedded in the basic medium SH+agar 10g L -1 + sucrose 40g·L -1 , pH 5.6, autoclaved at 125°C for 30min. Light culture at 24-26°C, light for 12 hours a day, light intensity 2200-2400lx;

[0078] SH basic formula ingredients are as follows: Potassium nitrate 3.5g L -1 , magnesium sulfate 0.29g L -1 , ammonium dihydrogen phosphate 0.46g L -1 , calcium chloride 1...

Embodiment 3

[0103] Example 3: Induction of callus and Agrobacterium-mediated high-efficiency transfection of "Red Lover" carnation

[0104] Step 1 Seed germination aseptic seedling stage: Rinse the seeds in sterile water for 3 times, pour out the sterile water, add 75% ethanol, shake for 30 seconds, pour out the ethanol, wash with sterile water for 3 times, pour out the sterile water , add 0.1% mercuric chloride, soak for 5 minutes, pour out the mercuric chloride and recycle, wash the seeds 8 times with sterile water, put the seeds on the filter paper and blow dry. Expose half of the seeds to the air, and inoculate them half-embedded in the basal medium (SH basal formula + agar 12g L -1 + sucrose 50g·L -1 , pH 5.6, autoclaved at 125°C for 20min. ) at 22-26°C for light culture, 12 hours of light per day, and light intensity of 2500lx;

[0105] SH basic formula ingredients are as follows: Potassium nitrate 2.5g L -1 , magnesium sulfate 0.2g·L -1 , ammonium dihydrogen phosphate 0.3g·L -1...

technical field

[0001] The invention belongs to the technical field of plant transformation mediated by Agrobacterium, in particular to a method for establishing a high-efficiency transfection system mediated by Agrobacterium carnation. Background technique

[0002] Carnation (Dianthus caryophyllus) is a model of beauty and elegance with exquisite body, colorful and elegant, dignified and generous, and fragrant and quiet. People often use carnations as gifts for their mothers, and use them to express their love and blessings to their mothers. With the rise of Mother's Day, carnations have become the most popular flower in the world. Carnation is the main export species of Kenya and the largest export flower species of Colombia in the Americas. It is widely cultivated in Japan, South Korea, Malaysia and other countries in Asia. In Europe, countries such as Germany, Hungary, Italy, Poland, Spain, Turkey, the United Kingdom and the Netherlands also have a large scale of culti...

Examples

Embodiment 1

[0051] Example 1: Callus induction and Agrobacterium-mediated high-efficiency transfection of "Xuewu" carnation

[0052] Step 1 Seed germination aseptic seedling stage: Rinse the seeds in sterile water for 3 times, pour out the sterile water, add 75% ethanol, shake for 30 seconds, pour out the ethanol, wash with sterile water for 3 times, pour out the sterile water , add 0.1% mercuric chloride, soak for 5 minutes, pour out the mercuric chloride and recycle, wash the seeds 8 times with sterile water, put the seeds on the filter paper and blow dry. Expose half of the seeds to the air, and half-embedded inoculated on the base medium SH+agar 8g L -1 + sucrose 20g·L -1 , pH 5.8, autoclaved at 121°C for 20min. Light culture at 22-24°C, light for 12 hours a day, light intensity 1800-2000lx;

[0053] SH basic formula is: Potassium nitrate 2.5g L -1 , magnesium sulfate 0.195g L -1 , ammonium dihydrogen phosphate 0.3g·L -1 , calcium chloride 151mg·L -1 , Glycine 2mg·L -1 , inosi...

Embodiment 2

[0076] Example 2: Induction of callus and Agrobacterium-mediated high-efficiency transfection of "Snow White" carnation

[0077] Step 1 Seed germination aseptic seedling stage: Rinse the seeds in sterile water for 3 times, pour out the sterile water, add 75% ethanol, shake for 30 seconds, pour out the ethanol, wash with sterile water for 3 times, pour out the sterile water , add 0.1% mercuric chloride, soak for 5 minutes, pour out the mercuric chloride and recycle, wash the seeds 8 times with sterile water, put the seeds on the filter paper and blow dry. Expose half of the seeds to the air, and inoculate them half-embedded in the basic medium SH+agar 10g L -1 + sucrose 40g·L -1 , pH 5.6, autoclaved at 125°C for 30min. Light culture at 24-26°C, light for 12 hours a day, light intensity 2200-2400lx;

[0078] SH basic formula ingredients are as follows: Potassium nitrate 3.5g L -1 , magnesium sulfate 0.29g L -1 , ammonium dihydrogen phosphate 0.46g L -1 , calcium chloride 1...

Embodiment 3

[0103] Example 3: Induction of callus and Agrobacterium-mediated high-efficiency transfection of "Red Lover" carnation

[0104] Step 1 Seed germination aseptic seedling stage: Rinse the seeds in sterile water for 3 times, pour out the sterile water, add 75% ethanol, shake for 30 seconds, pour out the ethanol, wash with sterile water for 3 times, pour out the sterile water , add 0.1% mercuric chloride, soak for 5 minutes, pour out the mercuric chloride and recycle, wash the seeds 8 times with sterile water, put the seeds on the filter paper and blow dry. Expose half of the seeds to the air, and inoculate them half-embedded in the basal medium (SH basal formula + agar 12g L -1 + sucrose 50g·L -1 , pH 5.6, autoclaved at 125°C for 20min. ) at 22-26°C for light culture, 12 hours of light per day, and light intensity of 2500lx;

[0105] SH basic formula ingredients are as follows: Potassium nitrate 2.5g L -1 , magnesium sulfate 0.2g·L -1 , ammonium dihydrogen phosphate 0.3g·L -1...

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