WO2012139416A1

WO2012139416A1 - Method of ultrasonic-aided agrobacterium mediated gene transformation in plant germinated seed

Info

Publication number: WO2012139416A1
Application number: PCT/CN2012/000307
Authority: WO
Inventors: 孙毅; 杨利艳; 杜建中; 郝曜山; 王亦学; 张丽君; 王铭; 于纪珍; 张婷婷
Original assignee: 山西省农业科学院生物技术研究中心
Priority date: 2011-04-15
Filing date: 2012-03-12
Publication date: 2012-10-18
Also published as: CN102212553A

Abstract

Provided is a method ofultrasonic-aided Agrobacterium mediated gene transformation in plant germinated seeds, including the following steps: with the plant germinated seeds as receptor and an Agrobacterium Ti plasmid carrying an exogenous gene fragment as a gene vector, scarring a growth site of the plant germinated seed receptor and treating with ultrasonic in the process of seed germination, co-culturing with Agrobacterium; after the exogenous gene is transferred to a genome of the receptor, directly screening the seeds or seedling, and performing PCR amplification and Southem hybridization on DNA of the plant leaves, to further determine the transformed plant. In the method, the plant germinated seeds are directly used as the receptor for Agrobacterium infection, without tissue culturing and plant regeneration.

Description

Ultrasound-assisted Agrobacterium-mediated transformation of plant germination seed genes

Technical field

The present invention relates to a method for transforming a plant gene, and more particularly to a novel method for using a ultrasonic treatment to treat scratches and Agrobacterium-mediated direct transformation of plant germinated seeds without the need for a tissue culture process.

technical background

In the Chinese patent No. ZL 01104185. 4, a transgenic method for directly transforming a maize germination embryo with Agrobacterium is disclosed, which is a method for genetically transforming a plant germinated seed by Agrobacterium, ie, The plant germinating seed is used as the acceptor material, and the Agrobacterium Ti plasmid carrying the foreign gene fragment is used as the gene donor, and co-cultured with the Agrobacterium during the seed germination process, so that the foreign gene fragment of the donor is transferred to the recipient. In the body genome. The method uses plant germinating seeds as a receptor to avoid the cumbersome and highly demanding plant tissue culture process. At the same time, using the seed as a receptor, the periodicity of the transformed plants and seeds obtained is greatly shortened, and the seeds are conveniently taken. Limited by season. However, the main disadvantage of this method is that the conversion rate is low, only about 0.6%, and it is easy to form a chimera.

In Chinese invention patents ZL 91103622. 9 and ZL 97111924. 4, Xu Ning et al. disclose a method for genetically transforming plant tissue blocks by ultrasonic waves. The plant tissue blocks they use are leaves, callus or immature embryos, and tissue culture processes are still required to obtain intact transformed plants and seeds. Sun Yi et al. proposed a method for ultrasonically treating pollen-induced gene transfer (Chinese patent ZL9912115. 2 ). In the method, the fresh pollen of the plant is placed in a container containing the exogenous DNA buffer, and the solution is ultrasonically treated to introduce the foreign gene into the plant pollen under the action of pulsed ultrasonic waves. All of the above inventions have shown that the use of ultrasonic treatment can make plants competent and facilitate the entry of foreign DNA. In these inventions, all used are plasmid DNA which enters plant cells by osmosis.

Summary of the invention

The object of the present invention is to overcome the above-mentioned deficiencies of the prior art, and provide an ultrasonic assisted Agrobacterium-mediated plant germination seed gene transformation method directly using plant germination seeds as Agrobacterium infection receptors without tissue culture and plant regeneration process. .

The specific technical scheme of the present invention is as follows: the germinated seed subjected to ultrasonic treatment after scratching the germinated young embryo is used as a receptor, and the Agrobacterium Ti plasmid carrying the foreign gene fragment is used as a gene carrier, and is co-produced with the Agrobacterium during plant seed germination. The recombinant gene is transferred to the recipient genome, and the transformant is further determined by PCR amplification and Southern hybridization of the leaf DNA of the plant by direct screening of the seed or seedling. In order to facilitate the entry of the Agrobacterium Ti plasmid into the recipient cell, the recipient plant germinating seed growth site is scratched and ultrasonicated, and then co-cultured with Agrobacterium, and the plant germinating seed growth site is scratched and immediately subjected to ultrasonic treatment. Make the apical meristem cells competent. The ultrasonic power used is 100-900 W, the frequency is 10 sec/time, and the number of treatments is 1-10 times. The embryo is the embryonic form of the plant. The apical meristem cells of the embryo have the ability to divide vigorously. When the growth point of the germinated seed embryo is scratched with a blade, it is too deep to damage the growth site, and the cell division ability is even decreased. Death; if the scratch is too shallow, the growth site is not injured, and the cells are difficult to be infected by Agrobacterium, so the scratch should be moderate. The depth of scratch varies slightly depending on the crop species and variety, generally 1-3 诵, and the scratch depth in the corn inbred line Chang 7-2 is about 2 s. (More specific). At the same time, on the basis of artificial scratching on the growth point of the germinated seed, ultrasonic treatment can make the growth point cells feel the state, further increase the conversion rate and reduce the formation of the chimera.

According to previous studies, dicotyledonous plants that are easily transformed by Agrobacterium usually secrete a phenolic substance, acetosyringone, at the site of infection, which activates the Vir gene on the Ti plasmid of Agrobacterium. Agrobacterium infection and transformation of receptors. Therefore, when the receptor is a leaf bud of the Agrobacterium host plant, it can be directly infected and transformed by Agrobacterium; when the receptor is a non-Agrobacterium host or a monocotyledonous leaf bud, a certain amount of acetyl clove can be added to the Agrobacterium suspension. Ketones act as inducers of Agrobacterium infection. In the present invention, the germinating seeds of the embryos and the ultrasonicated plants are infested and co-cultured by Agrobacterium; the co-cultivation of the germinated seeds and the Agrobacterium is carried out in agrobacterium liquid having a concentration of 106-108 cfu/ml, A final concentration of 10-200 umol/L acetosyringone was added to the bacterial solution.

After the germinated seeds are co-cultured with Agrobacterium, they can be directly sown in the seedbed, and the germinated seeds after co-culture (whether or not sifted) are sown in the seedbed to continue the screening. Or preliminary screening in an aqueous solution containing a screening agent.

The screening method may be based on the selection marker gene carried by the foreign gene vector using a corresponding screening agent, or using a molecular detection method such as PCR amplification or Southern hybridization. The plants with positive detection were selfed or crossed to obtain transgenic TO seeds, and the obtained TO transgenic seeds were sown to obtain T1 transgenic plants, and then self-crossing and molecular detection were continued until a stable homozygous transgene was obtained. Crossing or variety. The obtained inbred lines or varieties can be used as a parent for formulating hybrids or as a parent for cross breeding.

The present invention acts as an Agrobacterium infection receptor by scratching and ultrasonically treating the growth point portion of the germinated seed. After agrobacterium is co-cultured with the treated germinated seeds for a period of time, the seeds are then directly screened without tissue culture and plant regeneration. Ultrasonic treatment can also make the immature embryonic meristem cells in the sensation state in a state of sensation, which is beneficial to Agrobacterium infection, and is simpler, faster and more economical than other plant gene transformation methods.

In the traditional Agrobacterium-mediated gene transformation, untransformed cells can be eliminated by adding a screening marker to the culture medium, and the present invention is inspired by the seed and Agrobacterium co-culture for 48 hours, and then placed in a certain concentration. Screen the solution in the screening agent and then seed. The experiment confirmed that the foreign gene has been integrated into the recipient plant genome after Agrobacterium tumefaciens-mediated plant germinal seed gene transformation. The use of plant germination seeds as a receptor system for Agrobacterium-mediated transformation of foreign genes avoids the strict and cumbersome tissue culture process required by the traditional Agrobacterium co-culture method, shortens the experimental cycle, and proceeds during seed germination. Screening can effectively eliminate the number of unconverted seedlings, reduce the workload of post-testing, and save land. The invention does not require expensive instruments and complicated operation techniques, and utilizes plant germinating seeds as receptors, . It is easy to take and is not subject to seasonal restrictions. This method is easy to operate and can handle a large number of seeds at a time. The germinated seeds of corn, sorghum and wheat have been genetically transformed by the present invention.

DRAWINGS

Figure 1 is a physical map of the plasmid CrylAc;

Fig. 2 is a view showing a scratched portion after longitudinally scratching the germinated seed in the direction of growth of the germ.

In the figure: 1, seed coat or peel, 2, endosperm, 3, coleoptile, 4, germ, 5, cotyledon, 6, radicle, 7, radicle sheath.

detailed description

Example 1. Basic conversion method:

1) Agrobacterium LBA4404 carrying plasmid CrylAc was used as a foreign gene donor. The plasmid CrylAc carries the Bacillus thuringiensis venom AC gene and the plant selection marker gene bar, which confers resistance to the herbicide basta (the active ingredient is Phqsphiothricin or PPT). This gene was constructed by Wang Guoying, a researcher at the Crop Science Institute of the Chinese Academy of Agricultural Sciences. The physical map of the plasmid CrylAc is shown in Figure 1.

2) The maize (Zea mays L.) inbred lines Chang 7-2 and Zheng 58 , the sorghum restorer line Jinci 5, and the wheat variety Jinza 239 were used as acceptor materials. ,

3) Conversion operation process: First, the test seeds are rinsed in tap water, placed in 75% ethanol for 30 seconds, then rinsed in sterile water with 3 times in a clean bench and placed in glass container sterile water. (The height of the water is slightly above the seed), cover with autoclaved paper or parafilm, and place at room temperature (20-26 °C) on a shaker (100-200 rpm / 'min) 24-48 hour. After the germ part of the seed (pronounced "white"), the germinated seed was longitudinally scratched along the direction of growth of the germ with a scalpel (see Fig. 2 for the scratched part) and immediately subjected to ultrasonic treatment. Ultrasonic treatments have strengths of 300, 600 and 900W.

Agrobacterium single colonies containing the above genes were picked from LB plates containing 50 mg/L kanamycin (Km) and 20 mg/L rifampicin (rif) and inoculated in 10 mL containing 50 mg/L Km and 20 mg. /L rif in LB medium, 28 ° C, 180 r / min overnight culture to mid-turn (0D600 about 0. 4-0. 6) and add a final concentration of 100 μ mol / L acetosyringone. Add the above 50% and 50 mL of Agrobacterium liquid containing 10 ⁶ - 10 ⁸ cfu/mL to the germinated seed which was scratched and sonicated, and shake culture at 120-150 r/min. After -12 hours, the excess bacterial solution was decanted, and an equal amount of distilled water was added thereto, and placed on a shaker to continue the shaking co-culture. In this experiment, a treatment of adding 0.01% of the screening agent basta to the culture solution was also provided. The illumination was 12 hours and the temperature was maintained at 28 °C. After 2 days of co-cultivation, the germinated seeds were sown in the sand for screening culture, and sprayed onto the seedling leaves with 0.1% of the herbicide basta as a screening agent in a week or so. After 1-3 weeks of selection, healthy and well-developed basta-resistant plants were moved into the field. Normal growth management of seedlings transplanted into the field, self-crossing when flowering, and in grain Harvest when mature.

Example 2. Molecular detection of transgenic plants:

Molecular detection of transgenic plants was carried out according to the Molecular Cloning Manual:

1) In this experiment, total plant DNA was extracted by CTAB (hexadecanoyltriethylammonium bromide) method, and DNA extraction results and DNA quantification were performed by agarose gel electrophoresis. According to the nucleotide sequences of Cry lAc and bar genes, primers were designed by using 5' and 3' 20 bp nucleotide pairs for PCR amplification. The primer sequence and its amplified fragment size are as follows:

Cry lAc gene

Upstream primer: 5 ' -CTGACCGTGACCGTGCTG-3'

Downstream primer: 5 ' -TGGTGCCGTAGGCGAACT-3' .

Amplified fragment size: 500bp

Bar gene

Upstream primer: 5 ' - GTCTGCACCATCGTCAACC -3'

Downstream primer: 5, -ACTCGGCCGTCCAGTCGTA -3' .

Amplified fragment size: 201bp

The primers were synthesized by Shanghai Shenggong Bioengineering Co., Ltd. The PCR amplification was carried out using Dalian Ta Bioscience's 'TaKaRaTaqTM kit and PTC-200 type PCR machine. The plants obtained by transformation treatment and resistance screening were taken from leaves, and total DNA was extracted and PCR amplified. The amplification procedure is adjusted based on the primer sequence.

2) Southern hybridization: In order to further confirm the reliability of the PCR amplification results, the total DNA extracted from the positive PCR samples was randomly extracted and subjected to Southern hybridization. The plasmid DNA was used as a positive control, and the untransformed leaf DNA was a negative control. The results indicate that the foreign gene has indeed been integrated into the maize genome.

Example 3. Genetic transformation of maize:

In order to investigate the effects of various factors on the conversion rate, experiments were carried out including three factors including scratched germinated seeds, ultrasonic treatment, and whether or not a screening agent was added to the culture solution after 48 hours of co-culture. The ultrasonic treatment also included three intensity levels (300W, 600W and 900W), and genetic transformation of the maize inbred line Chang 7-2, with 4 replicates per treatment and 500 seeds per germination. A total of 1333 strains of basta resistant plants were screened for all treatments. After transplanting the plants screened by basta into the field, about 80% of the seedlings can form seedlings normally. Most of the plants showed normal behavior at the time of flowering, and only a small number of plants showed variability in flowering period and inability to loose powder. Molecular detection of leaves and extracted total DNA of basta-resistant strains was carried out according to the method of Example 2 to determine the conversion rate, and a total of 129 transformants were obtained.

The screening and molecular test results are shown in Table 1.

Table 1 Anti-basta screening and PCR detection results after maize seed gene transformation treatment Basta screens the number of surviving plants and the number of transformants (average conversion rate (transformed number of scratched agricultural sieves)

Treatment (average number SD)* number of soil SD)* 1 basta screening survival germination ultrasonic rod selection

Number of plants)% (average number of bacteria)

Soil SD)*

CK1 + ― + ― 28.00±8.83CD 2±0.82BC 7.41 ±3.34BC

CK2 + ― ― ― 38.75 ±14.38ABC 0±0C 0±0C

C 3 ― ― + ― ' 3.4.25 ±13.90 BC 0±0C 0±0C

C 4 ― ― ― ― 42.50±6.83 AB 0±0C 0±0C

1 + 300 + ― 40.75 ± 3.20ABC 3.25±2.22AB 21.35 ± 18.7A

2 + 600 + ― 33.00±2.38BC 5.0 ±2.58A 28.09±12· 87Α

3 + 900 + ― 31.00±2.99BC 4.5±1.91A 15·99±8.37ΑΒ

4 + 300 + + 15.25±3.20D 3.5 ±2.08AB 25.29±12.72Α

5 + 600 + + 17.50±2.38D 5 soil 0.82A 29.30±4.24Α

6 + 900 + + 28.25 ± 2.99 CD 5 ± 3.46A 17.83 ± 15.37ΑΒ

7 ― 300 + ― 49.50±10.44A 0.5±0.58C 1.07±1.25C

8 ― 600 + ― 44.00 ±2.94 AB 0±0C 0±0C

9 ― 900 + ― 41.75±12.89ABC 0.5±0.58C 1.4±1.05C

600/20

+ 32.25 ±6.60BC 5±1.41A 16.61 ±9.23ΑΒ

10 +

Times, 10 seconds

Treatment 1333 129 (32.25X4) 9.7%

Total

* Statistical analysis was performed on the experimental data using SPSS11.5 software. The variance analysis was performed using one way AN0VA, and the different letters indicated significant differences at the 0.05 level.

According to the results of PCR amplification and Southern hybridization detection, the maize inbred line is genetically transformed by the method of the present invention, and the foreign gene can be introduced into the recipient plant. It is better to treat scratched germinated seeds with 600W and 900W in various treatments, and to add acetosyringone to Agrobacterium liquid. It is better to treat all the seeds as the base, and an average conversion rate of about 1% can be obtained. Add 2.5 times (5/2) of the ultrasonic treatment. If the number of viable strains was screened by basta, the total conversion was '9.7% (129/1333). If the germinating seed is not scratched, only the intensity is 300-900W ultrasonic treatment (treatment 7-treatment 9), and the number of surviving plants is basta, the conversion rate is only 0-0.12%; only the germinating embryo is scratched without ultrasonic The conversion rate of the treatment was 7.41%, (CK1) but the difference between the treatment with the super-wave treatment only did not reach a significant level; after the seed was scratched, the ultrasonic treatment was performed (treatment 1 - treatment 6, and treatment 10). The conversion rate has increased dramatically to 15.99-28.09%. Whether or not the screening agent was added to the co-culture solution after co-cultivation for two days had no significant effect on the number of transformants obtained, but the number of false-positive strains was reduced, and the workload of post-sampling and DNA detection analysis was slightly reduced. Although there was no statistically significant difference between the number of transformants and the conversion between different ultrasonic intensity treatments, the higher intensity ultrasonic treatment (600 and 900 W) obtained a lower number of transformants (300 W). high. I Using this method, we have obtained anti-basta transgenic T1 maize seeds, indicating that the introduced foreign genes can be inherited. Example 4. Wheat genetic transformation:

Genetic transformation was also carried out in a similar manner on wheat (Jing Zuo 239), which treated 2000 seeds per treatment and 1000 seeds in control. After the transformation treatment, the seedlings were screened twice with 0.1% basta, and 181 plants resistant to the herbicide were obtained. Molecular testing of these herbicide-tolerant plants resulted in a number of transgenic plants (see Table 2).

* Conversion rate = (converted number / number of processed seeds) X 100%

As can be seen from Table 2, although the conversion rate was very low (0.12%), except for one treatment (500 W), the embryos were scratched and treated with ultrasonic waves to obtain transformants. Since there were no repeats and the number of transformants obtained was small, we did not perform a statistical analysis of the data. The reason for the low conversion rate of wheat and the unstable effect may be related to the fact that the seeds and embryos are small, and it is difficult to find the cells at the growth point during the scratch treatment. When the basta resistant strain was used as the base, the transformation rate was 9. 4% (17/181).

Example 5. Sorghum conversion results:

The conversion of the sorghum restoration system Jinci No. 5 was carried out. Since there was no duplication, no statistical analysis was performed on the data. However, as far as the existing results are concerned, except for one treatment (treatment 3-2), the rest of the ultrasonic treatment from 300W to 900W can obtain a certain amount of transformants, although the conversion rate based on the number of emergences is high. The conversion rate obtained by intensity treatment is slightly higher, but due to the small number of cases, statistically significant conclusions cannot be drawn. The addition of acetosyringone does not seem to have a major effect on the conversion of sorghum, and whether this is related to the high phenolic content of sorghum itself remains to be further studied. The conversion rate of all the treatments with the application of the ultrasonic wave is higher than that of the control without the treatment, which indicates that the ultrasonic treatment of the scratched germination embryo can greatly increase the conversion rate (average from 3.6% to 8.3%) ). The anti-basta transgenic T1 sorghum seed obtained by this method indicates that the introduced foreign gene can be inherited. The results of anti-basta screening and PCR detection after transgenic treatment of sorghum seed genes are shown in Table 3.

Table 3. Anti-basta screening and PCR detection results after sorghum seed gene transformation treatment Ultrasonic strong agricultural bar emergence number / emergence transformant number / transfer treatment acetyl syringone seed number

Rate (%) rate (%) *

3-1 300w + + 600 110 (18.3) 8 (7.3)

3-2 600w + + 600 36 (6) 0 (0)

3-3 900w + + 600 65 (10.8) 6 (9.2)

4-1 300w + - 600 79 (13.2) 5 (6.3)

4-2 600w + - 600 83 (13.8) 8 (9.6) .

4-3 900w + - 600 59 (9.8) 6 (10.1) Ultrasound

Treatment 3600 432 (12) 36 (8.3)

- + + 600 56 (9.3) 2 (3.60

* Conversion rate = (number of transformants / number of viable strains in basta screening) χ 100%.

It should also be noted that the ultrasonic treatment in this experiment was carried out using a Ningbo Xinzhi JY92-IID cell pulverizer with a maximum power of 900W. This power can be increased without further damage to the germinated seeds, and the processing time can be further extended.

Claims

Rights request

1. An ultrasonic assisted Agrobacterium tumefaciens germination seed gene transformation method, characterized in that a plant germinating seed is used as a receptor, and an Agrobacterium Ti plasmid carrying a foreign gene fragment is used as a gene vector, and the seed germination process is accompanied by agriculture. Co-culture of the bacilli, transfer of the foreign gene to the recipient genome, and direct screening of the seed or seedling

The DNA was subjected to PCR amplification and Southern hybridization to further determine the transformant.

2. The method according to claim 1, wherein the Agrobacterium tumefaciens-mediated plant germination seed gene transformation method is characterized in that the growth site of the germinated seed of the recipient plant is subjected to scratching and ultrasonic treatment, and co-cultured with Agrobacterium; scratch depth It varies slightly depending on the crop species and variety. The general scratch depth is about 2-3 awake; the ultrasonic power used is 100-900W, the frequency is 10 seconds/time, and the number of treatments is 1-10 times.

3 . The ultrasonic assisted Agrobacterium tumefaciens-mediated plant germination seed gene transformation method according to claim 1 , wherein the germinated seeds of the embryos and the ultrasonically treated plants are infected and co-cultured by Agrobacterium; the germinated seeds and the vegetative plants Co-culture of the bacillus was carried out in Agrobacterium liquid at a concentration of 106-108 cfu/ml, and a final concentration of 10-200 umol/L acetosyringone was added to the bacterial solution.

4. The method according to claim 3, wherein the germinated seed is co-cultured with Agrobacterium, directly seeded in a seedbed, or placed in an aqueous solution containing a screening agent. Conduct a preliminary screening.

The method according to claim 4, wherein the germinated seed after co-cultivation is sown in a seedbed to continue screening, and the screening method is carried according to the foreign gene carrier. The selectable marker gene uses a corresponding screening agent, or a molecular detection method such as PCR amplification or Southern hybridization.

The method according to claim 5, wherein the Agrobacterium tumefaciens-mediated plant germination seed gene transformation method is characterized in that the positively-detected plants are self-crossed or hybridized to obtain transgenic TO generation seeds.

7. The ultrasonic assisted Agrobacterium tumefaciens-mediated plant germinating seed gene transformation method according to claim 6, wherein the obtained TO-transgenic seed is sown, the T1 transgenic plant is obtained, and self-crossing and molecular detection are continued until Obtain a stable homozygous transgenic inbred line or variety.

The method according to claim 7, wherein the obtained inbred line or variety is used as a parent for formulating a hybrid or as a parent for cross breeding.