CN1179636C - Ultrasonic-assisted exogenous gene introduction method for soybean shoot tip transformation - Google Patents

Abstract

An ultrasonic-assisted exogenous gene introduction method for soybean shoot tip transformation. In the process of Agrobacterium-mediated BT gene transformation of soybean shoot tips, the ultrasonic-assisted method is used. After soybean seeds are germinated and pre-cultured, Agrobacterium infection is carried out Supplemented with ultrasonic infestation treatment, and then through co-cultivation, adventitious bud culture and transformed buds to induce rooting, to obtain seeds produced by transformed plants. The present invention uses ultrasonic waves to make tiny wounds on the explants, increases the contact area between the Agrobacterium and the explants, has a good effect in the process of introducing the BT gene, and obviously improves the infection efficiency of the Agrobacterium, effectively Increased the number of germination and the number of transformed seedlings.

Description

Ultrasonic-assisted exogenous gene introduction method for soybean shoot tip transformation

Technical field:

The invention relates to a method for exogenous gene introduction assisted by ultrasound-assisted transformation of soybean stem tips, which is an operation method for introducing exogenous genes into soybeans by using ultrasound as an auxiliary means, thereby improving the genetic traits of soybeans, belonging to biotechnology and modern agriculture technology field.

Background technique:

The success of plant gene transformation depends on whether it has a good receptor system, that is, whether it has strong plant regeneration ability and high-frequency transformation rate, ("Principles and Technologies of Plant Genetic Engineering" edited by Wang Guanlin, Technology Press). At present, the methods of genetic modification are mainly divided into two categories: 1 direct method, for example, pollen tube passage method, microinjection method, electric shock method, PEG method and gene gun method, 2 vector method, for example, Agrobacterium-mediated method and phage as carrier method wait. Among them, the Agrobacterium-mediated method plays an important role in plant transgenesis. Although some people have successively selected cotyledon nodes, immature embryos, etc. as explants for transformation, but the transformation efficiency is not high (0.6%). Bechtold used the vacuum infiltration technique to inoculate Arabidopsis plants with Agrobacterium to obtain a large number of mutants (Bechtold 1993.life sciences 316:1194-99). But the application of this technology on soybean has not been reported. Therefore, the main problem at present is to improve the conversion efficiency of soybean.

Invention content:

The purpose of the present invention is to overcome the deficiencies of the prior art, and provide a soybean stem tip transformation ultrasonic-assisted exogenous gene introduction method, so that soybeans have more germination, strong ability to regenerate seedlings, and improve soybean transformation rate.

In order to achieve such a purpose, the present invention adopts an ultrasonic-assisted method in the process of Agrobacterium-mediated BT gene transformation to soybean shoot tips. After soybean seeds are germinated and pre-cultivated, they are assisted by ultrasonic infestation during Agrobacterium infection. , so that the seeds are in the process of the airization effect of a certain selection pressure ultrasonic, the air bubbles are closed to form an instantaneous high pressure, and the continuous high pressure is like a series of small "explosions" that continuously impact the surface of the material, causing many micro-damages and avoiding the wounds cut with a knife in the past Excessive damage to explants also increases the contact area between Agrobacterium and explants, improving the infection efficiency of Agrobacterium. After cocultivation, adventitious bud culture and transformed bud induction rooting, the seeds produced by the transformed plant are obtained.

Method of the present invention comprises following specific steps:

1. Take soybean seeds and wash them repeatedly with tap water, soak them in 70% alcohol for 0.5-1 minute, transfer them to saturated sodium hypochlorite solution and soak them for 15 minutes, rinse them with sterile water for 4-5 times, and then place the seeds in sterile water. Soak in bacterial water for 18-24 hours to germinate the seeds.

2. Peel off the seed coat, remove the cotyledon, remove two original leaves under a dissecting microscope, expose the top meristem area, take out the shoot tip of about 0.5 cm from it, and place it in MSB (Murashige and Skoog (MS) medium Inorganic components + organic components in Gamborg (B5) medium) + 6-BA 3.0mg/L medium, pre-cultivated for 24 hours.

3. Take out the pre-cultured shoot tip, place it in a 100ml Erlenmeyer flask, add the prepared Agrobacterium liquid (OD600nm≈0.5), and immerse the Erlenmeyer flask in the center of an ultrasonic washer (power 500W, working frequency 50kHz). Ultrasonic infection treatment for 15 to 20 minutes.

4. After the infection is complete, take out the material, dry it with sterile filter paper, and put it into MSB+6-BA 1.0mg/L+IBA (indolebutyric acid) 0.2mg/L (PH5.8) medium to avoid light Cultured for 3 days.

5. Transfer to MSB+6-BA 0.5mg/L+cb (carbenicillin) 500mg/L (PH 5.8 liquid) for continuous culture for 3 days, and change fresh medium every day.

6. Transfer to MSB+6-BA (6-benzyladenine) 0.5mg/L+cb 500mg/L+KM (kanamycin sulfate) 80mg/L (PH5.8 solid), transfer every 2 weeks Once, until the adventitious buds appear and elongate to 2-3cm.

7. Cut off the adventitious buds on the explants and place them on 1/2MSB (the organic components in the inorganic components in the MS medium are halved+B5 medium)+KM50mg/L (PH5.8) medium, carry out The transformed shoots are induced to take root for 15-20 days, and then transplanted into large pots until the seeds produced by the transformed plants are obtained.

The present invention uses ultrasonic waves to make tiny wounds on explants, increases the contact area between Agrobacterium and explants, and has a good effect in the process of introducing BT genes, compared with the method mediated by ordinary Agrobacterium , significantly improved the infection efficiency of Agrobacterium, effectively increased the number of germination and the number of transformed seedlings.

Detailed ways:

The technical solutions of the present invention will be further described below in conjunction with specific embodiments.

Example 1

The experimental variety is Dongnong 43, 60 grains.

Soybean seeds are washed repeatedly with tap water, soaked in 70% alcohol for 0.5 minutes, then soaked in saturated sodium hypochlorite solution for 15 minutes, washed with sterile water for 4 times, and then soaked in sterile water for 18 hours. Peel off the seed coat, remove the cotyledon, remove two original leaves under a dissecting microscope, expose the top meristem area, take out the shoot tip of about 0.5cm from it, and place it on MSB+6-BA 3.0mg/L medium, Pre-cultivate for 24 hours to germinate the seeds.

Take out the pre-cultured shoot tip, place it in a 100ml Erlenmeyer flask, add the prepared Agrobacterium liquid (OD _600nm ≈ 0.5), immerse the Erlenmeyer flask in the center of an ultrasonic washer (power 500W, working frequency 50kHz), and ultrasonically Infection treatment for 20 minutes.

After the infection was complete, the materials were taken out, blotted dry with sterile filter paper, and placed in MSB+6-BA 1.0mg/L+IBA0.2mg/L (PH 5.8) medium for 3 days in the dark.

Transfer to MSB+6-BA 0.5mg/L+cb 500mg/L (PH5.8 liquid) for continuous culture for 3 days, and change fresh medium every day.

Transfer to MSB+6-BA 0.5mg/L+cb 500mg/L+KM80mg/L (PH5.8 solid), transfer once every 2 weeks, until adventitious buds appear and elongate to 2cm.

The adventitious buds on the explants were cut off and placed on 1/2MSB+KM50mg/L (PH5.8) medium, and the transformed buds were induced to take root for 15 days, and then transplanted to large pots until the seeds produced by the transformed plants were obtained.

34 soybean plants of East 43 transgenic soybean plants were obtained.

Example 2

The experimental variety is Jilin 27, 60 grains.

Soybean seeds are washed repeatedly with tap water, soaked in 70% alcohol for 1 minute, then soaked in saturated sodium hypochlorite solution for 15 minutes, washed with sterile water for 5 times, and then soaked in sterile water for 24 hours. Peel off the seed coat, remove the cotyledon, remove two original leaves under a dissecting microscope, expose the top meristem area, take out the shoot tip of about 0.5cm from it, and place it on MSB+6-BA 3.0mg/L medium, Pre-cultivate for 24 hours to germinate the seeds.

Take out the pre-cultured shoot tip, place it in a 100ml Erlenmeyer flask, add the prepared Agrobacterium liquid (OD _600nm ≈ 0.5), immerse the Erlenmeyer flask in the center of an ultrasonic washer (power 500W, working frequency 50kHz), and ultrasonically Infection treatment for 15 minutes.

After the infection was complete, the materials were taken out, blotted dry with sterile filter paper, and placed in MSB+6-BA 1.0mg/L+IBA0.2mg/L (PH5.8) culture medium for 3 days in the dark.

Transfer to MSB+6-BA 0.5mg/L+cb 500mg/L (PH5.8 liquid) for continuous culture for 3 days, and change fresh medium every day.

Transfer to MSB+6-BA 0.5mg/L+cb 500mg/L+KM80mg/L (PH5.8 solid), transfer once every 2 weeks, until adventitious buds appear and elongate to 3cm.

The adventitious buds on the explants were cut off and placed on 1/2MSB+KM50mg/L (PH5.8) medium, and the transformed buds were induced to take root for 20 days, and then transplanted to large pots until the seeds produced by the transformed plants were obtained.

Obtain 36 soybean plants of Jilin 27 transgenic with BT gene.

Example 3

The experimental variety is Hefeng 35, 60 capsules.

Soybean seeds were washed repeatedly with tap water, soaked in 70% alcohol for 0.7 minutes, then soaked in saturated sodium hypochlorite solution for 15 minutes, rinsed with sterile water for 5 times, and then soaked in sterile water for 19 hours. Peel off the seed coat, remove the cotyledon, remove two original leaves under a dissecting microscope, expose the top meristem area, take out the shoot tip of about 0.5cm from it, and place it on the MSB+6-BA3.0mg/L medium, Pre-cultivate for 24 hours to germinate the seeds.

Take out the pre-cultured shoot tip, place it in a 100ml Erlenmeyer flask, add the prepared Agrobacterium liquid (OD _600nm ≈ 0.5), immerse the Erlenmeyer flask in the center of an ultrasonic washer (power 500W, working frequency 50kHz), and ultrasonically The infection treatment was 18 minutes.

After the infection was complete, the materials were taken out, blotted dry with sterile filter paper, and placed in MSB+6-BA 1.0mg/L+IBA0.2mg/L (PH5.8) culture medium for 3 days in the dark.

Transfer to MSB+6-BA 0.5mg/L+cb 500mg/L (PH5.8 liquid) for continuous culture for 3 days, and change fresh medium every day.

Transfer to MSB+6-BA 0.5mg/L+cb500mg/L+KM80mg/L (pH5.8 solid), transfer once every 2 weeks until adventitious buds appear and elongate to 2cm.

The adventitious buds on the explants were cut off and placed on 1/2MSB+KM50mg/L (PH5.8) medium, and the transformed buds were induced to take root for 18 days, and then transplanted to large pots until the seeds produced by the transformed plants were obtained.

37 soybean plants of Hefeng No. 35 transgenic with BT gene were obtained.

The method of the invention is suitable for soybean transfection of various genes, and can quickly obtain high-frequency soybean transformation plants.

Claims (1)

1, a kind of exogenous gene introduction method assisted by ultrasonic wave of soybean stem tip transformation, is characterized in that comprising the steps: 1) Soybean seeds are rinsed and soaked in 70% alcohol for 0.5-1 minute, then soaked in saturated sodium hypochlorite solution for 15 minutes, rinsed with sterile water, and soaked in sterile water for 18-24 hours, so that seed sprouting; 2) Remove the seed coat, remove the cotyledon, remove two original leaves under a dissecting microscope, expose the top meristem area, take out the shoot tip of about 0.5cm from it, and place it on MSB+6-BA 3.0mg/L medium , pre-cultured for 24 hours; 3) Take out the pre-cultured stem tip, put it in a 100ml Erlenmeyer flask, add the prepared Agrobacterium liquid (OD _600nm ≈ 0.5), and immerse the Erlenmeyer flask in an ultrasonic washer with a power of 500W and a working frequency of 50kHz. Infection treatment for 15 to 20 minutes; 4) After the infection is complete, the material is taken out, blotted dry with sterile filter paper, and placed in MSB+6-BA 1.0mg/L+IBA0.2mg/L (PH5.8) culture medium for 3 days in the dark; 5) Transfer to MSB+6-BA 0.5mg/L+cb 500mg/L (PH5.8 liquid) for continuous culture for 3 days, and change fresh medium every day; 6) Transfer to MSB+6-BA 0.5mg/L+cb 500mg/L+KM80mg/L (PH5.8 solid), transfer once every 2 weeks, until adventitious buds appear and elongate to 2-3cm; 7) Cut off the adventitious buds on the explants and place them on 1/2MSB+KM50mg/L (PH5.8) medium to induce rooting for 15-20 days by transforming buds, and transplant them into large pots to obtain transformed plants. The seeds of which 1/2 MSB is halved the inorganic components in the MS medium + the organic components in the B5 medium.