Current status and prospects of transgenic research in major crops
1 Progress in transgenic research of major crops
1.1 Soybean Genetic Transformation Technology and Transgenic Research
Soybean is one of the most difficult crops in genetic engineering. The first reason is that soybean tissue culture is not mature enough. Although there are many reports that can be derived from soybean hypocotyl, cotyledonary nodes, cotyledons, leaves, young cotyledon, immature embryos, anthers, etc. Explants obtained regenerated plants, but the frequency was lower and the reproducibility was poorer. The second reason is that soybean is very sensitive to Agrobacterium. Once infected, it is difficult to regenerate plants from specific tissues or cells. Due to the importance of soybeans, researchers have been trying to create effective tissue culture and plant regeneration systems. In any case, the genetic transformation of soybeans has achieved great success. The earliest research on genetic transformation of soybeans used protoplasts as explants. In 1988, Hinchee et al. used Agrobacterium-mediated method to obtain soybean transgenic plants for the first time, introduced nptII gene and glyphosate resistant gene into soybean, and McCabe used gene gun method to obtain transgenic soybean plants with nptII gene. In 1989, Parrott et al. used particle bombardment to obtain soybean transgenic plants that transfused corn with a 15 kDa alcohol-soluble egg gene and nptII gene; Liu Bolin introduced the anti-attriz gene psbA from Solanum nigrum to soybean using a ovary microinjection method during the zygote phase. The chloroplast genome was obtained as a transgenic soybean. Sato et al. used gene gun method to obtain transformed soybean regenerated plants through embryogenesis through cell suspension lines; Christou et al. used the electroporation transformation method to obtain multiple transformed cell lines with stable expression of foreign genes. Finer et al. used gene guns to bombard soy embryoid body suspension cultures and used hygromycin as a selective agent to obtain a large number of regenerated plants. Huang Jianqiu et al. used a modified PEG-mediated method to transform GUS gene of soybean protoplasts and detected the stable expression of GUS gene. Stewart et al. Obtained fertile soybean plants with CryIA gene transfer; Monsanto, USA used gene gun bombardment method to transfer 5-enol-pyruvate oxalate-phosphate synthase (EPSPS) gene into soybean plants to obtain herbicide-resistance Genetically modified soybeans, Agrobacterium-mediated transformation of herbicide-tolerant Roundup genes into soybeans, development of Roundup Ready genetically modified soybeans, and industrialization of large areas; Wei et al. used PEG method to introduce foreign genes into soybean protoplasts. Transgenic plants. Xu Xiangling et al. used Agrobacterium-mediated method to transfer the soybean mosaic virus coat protein gene into soybean plants; Nan Xiangri et al. obtained transgenic Bt soybean plants. Zhang et al. found that in the cotyledonary node transformation system, using the bar gene and the corresponding selection agent glufosinate, the selection effect was significantly better than other marker genes. Xing et al. obtained a transgenic soybean plant without a selection marker by constructing a binary expression vector for the secondary T-DNA. Sato et al. used Agrobacterium-mediated method to transfer the Δ-fatty acid desaturase gene cloned from the sorghum to soybean plants, and obtained transgenic lines without selection markers. The content of γ-linolenic acid in seeds was significantly increased. . In addition, people are still trying to transfer the glucanase gene, ribosome inactivating protein gene and chitinase gene into soybeans to increase the resistance of soybean to fungal diseases and transfer the genes related to vitamin A synthesis into soybeans. Soybean nutritional quality, the ribulose 1,5 diphosphate carboxylase gene was transferred to soybeans to improve soybean photosynthetic efficiency and increase soybean yield.
Since the acquisition of the first case of genetically modified soybeans, there have been many successful reports, including industrialized herbicide-resistant transgenic soybeans. At present, the most commonly used method is still the Agrobacterium-mediated regeneration system of cotyledonary nodes, followed by the gene gun-mediated embryogenic suspension culture regeneration system. Other transformation methods need to be further improved, including pollen tube DNA uptake, Agrobacterium floret infiltration, and other soybean explant transformation systems mediated by Agrobacterium and gene guns. The most effective Agrobacterium strain for soybean transformation was EHA101. The explants were cotyledonary nodes. The regeneration pathway was the organogenesis pathway that induced cluster buds. The selection marker was bar gene and the selection agent was glufosinate. After obtaining resistant soybean regenerating plants, in addition to the commonly used methods of histochemical staining and molecular biology, it is also possible to rapidly detect transgenic soybeans using a leaf-sweeping herbicide.
1.2 Status of Maize Genetic Transformation Technology and Transgenic Research
Fromm et al. first carried out the maize genetic transformation work and used electroporation to induce maize protoplasts to absorb plasmid DNA carrying the chloramphenicol acetyltransferase gene. After the electroporation treatment, the expression of this gene in corn cells was detected, and the nptII gene was further transferred. Into the maize protoplasts, stable transformed resistant calli were obtained. Rhodes et al. used electroporation to transfer the nptII enzyme gene into maize protoplasts. The corn transgenic plants were obtained for the first time, but the transgenic plants were all sterile. Grimsly et al. constructed the maize streak virus gene on the Ti plasmid of Agrobacterium tumefaciens and used Agrobacterium to infect corn plants, resulting in symptoms of systemic infection. It was demonstrated for the first time that Agrobacterium could infect corn cells. Fromm et al. and Gordon-Kamm et al. transformed the bar gene, GUS gene and firefly luciferase gene into corn by gene gun-mediated method, and obtained transgenic plants. Ding Qunxing et al. used micro-glass needles to inject the corn ovary 10 to 20 hours after pollination, and transferred Bacillus thuringiensis (Bt) insecticidal protein gene into maize to obtain fertile transgenic plants. Wang Yingying and others used particle guns to bombard maize suspension cell lines, immature embryos and embryogenic calli, and transferred the Bt gene and bar gene to maize. Ishida et al. used Agrobacterium-mediated transformation of maize inbred A188 immature embryos to obtain a large number of transgenic plants and established a maize-mediated Agrobacterium-mediated transformation technology system. Zhang Hong et al. used ultrasound to transform maize and obtained transgenic plants. Zhang Rong et al. broadened the genotypes of Agrobacterium-transformed maize to conventional inbred ensembles3 and Zou 31. Zhang Yanzhen and others used Agrobacterium-mediated introduction of Bt toxin genes into maize elite inbred lines 340 and 4112. The average conversion rate reached 2.35%.
In summary, although methods used in corn transformation include electroporation, gene gun-mediated method, ultrasonic method, microinjection method, PEG method, and Agrobacterium-mediated method, gene gun-mediated method and Agrobacterium-mediated method. The guide method is the most successful application. About 18 days after pollination, immature embryos are commonly used receptor materials, which have strong regeneration ability and good transformation effect. For the Agrobacterium-mediated method, the maize genotypes suitable for transformation are still relatively limited. The suitable Agrobacterium strains are C58C1 and LBA4404, and the appropriate amount of acetosyringone (AS), proline, and glutamine are added to the co-culture medium. Amides and other substances are beneficial to Agrobacterium infecting immature embryos of maize and increasing transformation efficiency.
1.3 Status of Rice Genetic Transformation Technology and Transgenic Research
In the late 1980s, it was reported at home and abroad that PEG method and electric shock method were used to transform rice protoplasts to obtain rice transgenic plants. Due to the complexity of protoplast culture operations, protoplasts are used as transforming receptors, and the rate of transformation and regeneration is very low. In the early 1990s, people tried to apply the Agrobacterium transformation technology commonly used in dicotyledons to rice transgenic research. Li Baojian and others first used Agrobacterium to transform rice and detected the expression of exogenous genes at the callus level by adding phenolic compounds such as AS, vanillin, and gallic acid to Agrobacterium bacterium liquid and infection medium. And under the electron microscope, the adhesion and infection of Agrobacterium on rice cells were observed. Chan et al. used the immature embryos 10 to 12 days after flowering and pollination of rice as recipients, and obtained transgenic plants after infection with Agrobacterium, but also lacked molecular biological evidence. Li Yao et al. used Agrobacterium tumefaciens to transform adventitious buds of rice and obtained transformed plants with GUS expression but lacked molecular biological evidence. Hiei et al. used rice mature embryo calli and immature embryos as recipients to obtain more transgenic plants with rigorous molecular biology evidence. The influencing factors of Agrobacterium-mediated rice transformation were studied in detail. A more mature technology system for Agrobacterium transformation rice has opened the way for Agrobacterium-mediated transformation of monocotyledons. It also laid the foundation for the successful transformation of indica rice by Agrobacterium using Rashid et al.
Ye et al. introduced the three genes psy, crtI, and aphIV of the β-carotene synthesis pathway into rice, and developed a new line containing vitamin A. Eunpyo et al. (1989), Zhang Xianyin (2001) et al., Gao Yuefeng et al introduced the storage protein gene Glutein, globulin gene, and high lysine gene into rice, respectively, and the quality of the transgenic rice was improved to varying degrees. He Yingchun et al. introduced the chitinase gene into rice using the pollen tube pathway method, and obtained the progeny of the variation, and provided evidence of molecular hybridization. However, the conversion efficiency of this method is still relatively low, and the operation is also limited by the flowering season, and the screening workload for offspring is large. Xu Minghui and others successively used the gene gun method to transfer the chitinase-glucanase bivalent gene into rice. Compared with the recipient variety, the transgenic rice has significantly increased resistance to rice blast. Hossan et al. (1991) transferred three genes, pdcI, pdcII, and pdcIII, related to flood tolerance in rice, to rice and obtained transgenic plants. Murata Hirofaki et al. and Hiroshi Kazuko introduced the betaine biosynthetic enzyme genes codA and betA into rice to obtain transgenic plants with high salt tolerance.
The transformation of foreign insect-resistance genes is the most active research field in rice transgenic research. The foreign genes used include insecticidal crystal protein gene, protease inhibitor gene, lectin gene, neurotoxin gene, such as CryIA(b), CryIA. (c), cpTI, GNA, AaIT, etc. At present, China has established a technical system for the stable transformation of SCK insect-resistant genes into rice by Agrobacterium, with a conversion efficiency of more than 5%. Gatehouse et al. reported on the results of a study on the resistance to brown planthopper in rice of the G. avens lectin GNA gene.
Genetic breeding of bacterial blight resistance and breeding of genetically engineered herbicides are another hotspot in transgenic rice research. Tu et al. introduced the Xa21 gene into rice varieties such as IRF-2 by using an agrobacterium-mediated method, and the resistance of the transgenic rice to bacterial blight was significantly enhanced. Huang Da Nian and others used the gene gun method to introduce antimicrobial peptide genes into Jingyin 119 rice, and the transgenic plants were resistant to bacterial blight; the bar gene, which is resistant to herbicides, was introduced into the hybrid rice restorer line. Since the maintainer line does not contain the bar gene, The spraying of Basta herbicides at the seedling stage of hybrids not only kills weeds, but also removes false hybrids. Transgenic herbicide resistant rice has been field tested. The application of transgenic technology to breed new male sterile lines, improve stress resistance, prevent premature aging of rice and improve the quality of rice is ongoing.
The Agrobacterium-mediated rice genetic transformation system is very mature, widely used at home and abroad, and there is no strict genotypic specificity. The embryogenic calli and immature embryos are pre-cultured on AAM medium for 2 to 3 days. Embryos can be used as exogenous gene transfer acceptor materials, and their regeneration ability and transformation ability are ideal. Successful Agrobacterium strains include LBA4404 and A281. Gene gun-mediated rice genetic transformation system is relatively mature, and its practical application is also more extensive.
1.4 Status of Wheat Genetic Transformation Technology and Transgenic Research
Among the food crops, wheat belongs to the most difficult crops for genetic transformation. Coupled with the late start of transgenic research, the genetic engineering breeding process obviously lags behind other crops. With the advent of particle guns, the use of new marker genes and efficient promoters, wheat genetic research began to increase after 1991. Vasil et al. used the gene gun-mediated method to introduce the bar gene into wheat and obtained the world's first wheat transgenic plant. Weeks and others used gene gun-mediated method to introduce the GUS gene and bar gene into wheat, and established a technological system for the transformation of wheat by the gene gun method. In the following years, wheat transgenic studies basically rely on gene gun-mediated methods. Zhou et al. used gene gun-mediated method to introduce CP4 gene and GOX gene into wheat and obtained transgenic plants. Blechl et al. introduced high-molecular-weight glutenin subunit HMW-GS gene into wheat immature embryos and young panicles, and obtained stable transgenic plants. However, Agrobacterium-mediated method has always been a difficult problem for wheat genetic transformation. Cheng et al. used the Agrobacterium-mediated method to transfer the GUS gene and the nptII gene into wheat for the first time. The wheat transgenic plants were obtained, and the T0 generation-T2 generations of the transgenic plants were molecularly detected. Xia Guangmin and Ye Xingguo used Agrobacterium-mediated methods to transfer exogenous genes such as nptII and bar into wheat and obtained transgenic plants. Since 2001, the focus of transgenic wheat research has shifted from the establishment of transformation systems to functional gene transfer. Zhou et al. used Agrobacterium-mediated method to transfer the Roundup gene of herbicide-resistance to wheat variety Bobwhite and cultivated Roundup Ready wheat. Transgenic wheat showed strong resistance to herbicides. Production trials had been completed and industrialization was awaited. Xu Huijun and others used gene gun-mediated method to introduce Nib8 replicase gene into wheat. Xu Qiongfang et al. and Liang Hui et al. introduced the GNA gene into wheat using gene gun-mediated method. Transgenic wheat showed strong resistance to yellow mosaic virus disease and aphid. Sex, has entered the environmental release phase. The authors introduced GCE, Bcl and Rip genes into wheat using Agrobacterium-mediated method. The results of the intermediate experiment showed that the transgenic wheat showed some resistance to head blight.
In the report on the transgenic plants that have been obtained so far, the gene gun method accounts for about 90%, and the other methods only account for 10%, including Agrobacterium-mediated method, pollen tube channel method, and low-energy argon ion beam-mediated method. Due to the merits of Agrobacterium-mediated methods, people have been persistent in this research. In 2003, Khanna et al. constructed a super binary expression vector HK21 and added polyamine compounds to the medium. The transformation efficiency reached 1.2% to 3.9%. Hu et al. selected the herbicide-resistant gene EPSPS from Agrobacterium as the selection marker. Using glufosinate as a screening agent, the EPSPS gene was introduced into the wheat variety Bobwhite, and the transformation efficiency reached 4.3%. Cheng et al. believe that the drying of explants after Agrobacterium infection can significantly increase T-DNA translocation and transformation efficiency.
Most transgenic wheat plants use 13 to 14 days after pollination as the recipient material. Ubi, E35S promoters and screening markers such as bar, nptII and EPSPS are commonly used in the construction of expression vectors. Bialaphos and Glufosinate are commonly used as screening agents. G418 and Glyphosate et al. Successfully used Agrobacterium strains including ABI, Agl1, c58C1, LBA4404, and CP4. In conclusion, wheat transgene research involves more reporter genes or marker genes, fewer target genes, and most of the recipient genotypes used are Bobwhite. Therefore, broadening the range of receptors for wheat genetic transformation, perfecting the technical system for the transformation of wheat by Agrobacterium, and introducing some foreign genes that control disease resistance, high quality, stress resistance, and insect resistance into wheat are the focus of wheat genetic research in the future.
2 Industrialization of transgenic crops
2.1 Global cultivation of transgenic crops
Since the first GM plant was approved for field trials in 1986, 30 countries have ratified thousands of transgenic plants in field trials, involving more than 40 plant species. In 1994, GM tomatoes were approved for marketing in the United States. In 1995, GM cotton was approved for commercial production. GM rapeseed was approved for promotion in Daejeon in Canada. In 1996, GM corn began commercial cultivation in the United States. In 1996, the global GM crop planting area reached 1.7 million hectares. In 1999, GM soyabeans were approved for listing in the United States. The global acreage of GM crops grew to 44.2 million hectares in 2000, a 25-fold increase over 1996. In 2001, GM maize was planted in large areas in the United States. GM soybeans were grown in countries such as Brazil and Argentina. The area of ​​GM crops planted in the world increased to 81 million hectares in 2004, an increase of 19.7% over 2003, and an increase of nearly 47% over 1996. Times. So far, seven transgenic plants have been approved for commercial planting in 18 countries, including tomatoes, petunias, bell peppers, pumpkins, and papayas, in addition to major crops such as soybeans, corn, cotton, and rapeseed. Among them, the genetically modified soybean has the largest planting area, accounting for 60.0% of the total area of ​​the global transgenic plants, followed by genetically modified corn, transgenic cotton and genetically modified rapeseed. As far as the world is concerned, the United States is the country with the largest area of ​​genetically modified crops, accounting for 59.0% of the world's total genetically modified crops, followed by Argentina, Canada, Brazil, and China. The GM crops grown in the United States are mainly soybeans, corn, etc. The genetically modified crops grown in Argentina and Brazil are mainly soybeans, the transgenic crops grown in Canada are mainly rapeseed, and the genetically modified crops grown in China are mainly cotton. According to the type of genetically modified crops, 73.0% of the herbicide-tolerant herbicides were genetically modified soybeans and oilseed rape, and 18.0% were resistant to insects, which were mainly genetically modified corn and cotton. The type of anti-herbicide accounted for 9.0%. The main types were genetically modified and resistant to viruses. Less than 1.0%, mainly papaya.
2.2 Planting of Transgenic Crops in China
Under the support of the national "863" program and the "Special Project of Research and Industrialization of Transgenic Plants" and other scientific and technological projects, great progress has been made in the research of transgenic plants in China. So far, more than 1,000 cases of genetically modified plants have reported safety assessments, and nearly 800 cases have been approved. Of these, more than 450 were approved for intermediate trials, 200 were approved for environmental release, and more than 50 were approved for productive trials, including genetically modified rice and cotton. There are 30 plants such as corn, rape, potato, soybean, and wheat. Resistant storage tomatoes, ornamental petunias, ornamental petunias, boll cotton, insect-resistant cotton, anti-viral sweet peppers, and anti-viral tomatoes, and other transgenic plants are approved for commercial production. The largest genetically modified crop in China is insect-resistant cotton. In 2002, the planting area was 2 million hectares. In 2003, the planting area was 2.8 million hectares. In 2004, it reached 3.7 million hectares. However, compared with the international advanced level, there is still a certain gap between China's genetic research. The planting area of ​​genetically modified crops only accounts for 5% of the world's total. The genetically modified crops involved are mainly cotton.
2.3 Economic Benefits of Transgenic Crops
The promotion of GM crops has produced considerable economic benefits globally. It has not only significantly reduced the amount of pesticides used, effectively controlled pests and weeds, and reduced environmental and agricultural pollution, but also significantly increased crop yields and farmers' income. According to ISAAA estimates, the annual trade volume of GM crop seeds in the global market has increased from the initial 150 million US dollars to more than 3 billion US dollars in recent years. The US income from growing GM soybeans increased by about $50 per hectare, the yield from growing GM cotton increased by about $40 per hectare, and the yield from growing GM corn increased by more than $20 per hectare. From the perspective of planting genetically modified corn in the United States and Canada, the control effect of corn borer is very prominent. Compared with the non-transgenic control of the same variety, the insect-resistant corn has an average yield increase of 7% to 9%. The global promotion of Bt cotton alone has saved more than 33,000 tons of pesticides, which has reduced by about 40%. In 2001, 6 transgenic crops grown in the United States reduced the use of pesticides by more than 23,000 tons, which not only reduced production costs, but also protected surroundings. From 1996 to 2003, the total cultivated area of ​​insect-resistant cotton in China reached 4.8 million hectares. The market share of domestic insect-resistant cotton varieties increased from 10% in 1998 to 64.4% in 2002. The cotton farmers increased their income by more than 2 billion yuan each year. The average increase of more than 700 yuan in hectares.
3 Problems and Prospects of Crop Transgenic Research
3.1 Transformation Method Application and Conversion System Establishment
Agrobacterium-mediated method is the most widely used method of transgenic plants. In natural conditions, Agrobacterium infects only dicotyledonous plants and does not infect monocotyledonous plants. Therefore, early people thought that Agrobacterium-mediated transformation methods could not be used for monocots. Because Agrobacterium tumefaciens transformation has many advantages such as high transformation efficiency, clear exogenous inserts and single-copy integration, many researchers have been exploring the transformation of cereal grain crops with Agrobacterium. In recent years, although Agrobacterium-mediated transformation systems have been established in major food crops, the transformation effects have greatly depended on species, genotypes, explants, and other unknown factors. Especially for wheat and corn, the requirements for genotypes and explants are more stringent. Further increase the frequency of transformation of these two major crops and establish a highly efficient transformation system that is not genotype restricted.
The gene gun-mediated method was the most used in wheat, followed by maize and rice. However, the gene gun-mediated method has a relatively low conversion efficiency, the fragment size of the inserted foreign DNA is not clear, there are many multiple copy integration, gene silencing is easy to occur, can not introduce large fragments of DNA and other disadvantages, and the cost is high, the operation is more complicated. There are some limitations in practical applications.
The pollen tube pathway method has many successful cases in China. It has obtained transgenic plants such as wheat, cotton, corn, rice, and soybean. Some transgenic varieties have entered industrial production and have strong practicality. Pollen-mediated transformation is an extension of the pollen tube pathway method and has been successfully reported in some plants. However, the transgenic plants obtained by the pollen tube pathway method lacked strict molecular biological evidence and the theoretical basis was insufficient. It is necessary to strengthen the research in this area.
The establishment of a simple and efficient transformation system is of great significance for the research of genetic transformation of major crops. The in vitro transformation of Agrobacterium tumefaciens into flowering plants is very effective in Arabidopsis thaliana. Transgenic plants can be obtained by seedlings or seedlings spraying selective agents after they have matured. If this method can be successfully applied to the main crops, it will bypass the tissue culture and facilitate the popularization of transgenic technology.
3.2 Strategies for improving the conversion efficiency of major crops
Tissue culture and plant regeneration pathways are the basis of plant transgenic research. Among the major grass crops, the technical system for the transformation of rice by Agrobacterium is the most mature, which may be due to the fact that the transformation of rice mostly uses mature embryos as the acceptor material, which not only has strong regenerative ability, but also has convenient materials. The transgenic research of wheat, corn and other crops mostly uses immature embryos as the receptor material, has strong genotype specificity, establishes a high-frequency regeneration system for mature embryos, and screens genotypes sensitive to Agrobacterium, for improving crops such as wheat and corn. The conversion efficiency has a promoting effect. After nearly 2 years of research and exploration, we used Agrobacterium to transform the wheat mature embryo callus for the first time to obtain transgenic plants, which provided a new way to further increase the transformation efficiency of Agrobacterium.
Agrobacterium-plant cell signaling and interactions are key steps in achieving plant gene transfer. Monocotyledonous plants such as wheat, corn, and rice are insensitive to Agrobacterium, improving the external environment of the co-culture process, screening suitable Agrobacterium strains and receptor genotypes, etc. can promote the shear and transfer of T-DNA. At the same time, by transferring regulatory genes such as VirGN54D into Agrobacterium donors and binding protein genes such as VIP1 into plant receptors, collective expression of Vir genes can be induced, and T-DNA intercellular transfer levels and transport to the nucleus can be increased. Integration into chromosomes has great potential for improving Agrobacterium transformation efficiency. In addition, the construction of highly effective expression vector is not only conducive to the expression of foreign genes, but also conducive to the selection of transformed cells. Promoter tandem strategy and enhancer optimization can avoid the inefficient expression and silencing of foreign genes.
3.3 Functional gene transfer and application
From the first transgenic plants to the present, there have been reports of multiple gene transfer to various plants, involving antiviral, antifungal, insect-resistant, herbicide-resistant, drought-resistant, and improved quality traits. However, from the point of view of currently used transgenic plants, there are mainly two types of transgenic plants that are of practical application value. One is Bt-resistant insect-resistant plants, and the other is the herbicide resistant to roundup genes and EPSPS or bar genes. Plants, most of the other transgenic plants do not play their intended role in production practice because of the weak function of the transferred genes. Therefore, the excavation and cloning of genes with important utilization values ​​should be strengthened, and further transferred to major crops to increase agricultural output and farmers' income.
3.4 Detection and Functional Identification of Transgenic Plants
After the transgenic plants are obtained, strict molecular biological tests and scientific functional identification are made, which are conducive to the popularization of transgenic plants. Based on preliminary identification of transgenic plants such as PCR, ELISA, and leaf smear herbicides, Southern, Northern, and Western identifications were performed to detect the integration, transcription, and expression of functional genes. Especially for the transgenic plants obtained by the Agrobacterium-mediated method, due to the presence of Agrobacterium tumefaciens in the T0 plants, the PCR test results have a certain degree of false positives. Functional verification should be performed under strictly controlled conditions, setting up duplicates and controls.
3.5 Safe GM crop cultivation
In general, transgenic plants contain marker genes in addition to the gene of interest. The selection marker not only affects the safety evaluation of transgenic plants, but also has hidden dangers of biosafety and environmental safety. It needs to be excluded from the self-progenies or hybrids of transgenic plants. At present, the techniques for obtaining transgenic plants without selection markers include co-transformation, recombination and positioning systems, and MAT vector systems, among which the co-transformation method is most effective. The co-transformation method of the mixed plasmid gene gun bombardment has some defects. In addition to the target gene and the marker gene, the two carriers carry other unwanted DNA sequences. The two-stage T-DNA vector-mediated Agrobacterium-mediated co-transformation method is safe and reliable, and the efficiency of obtaining unlabeled transgenic plants is also high. The authors transformed soybeans by constructing a binary expression vector containing three T-DNAs (the target gene is located on the second T-DNA and the marker gene on the other T-DNA), and the frequency of the unmarked transgenic plants in the T1 generation was 7.6%. .
3.6 Crop Genetic Transformation - Molecular Design Breeding
Some of the main characteristics of plants are the inheritance of quantitative traits, such as yield, quality, stress resistance, etc. In particular, the metabolic pathways of some key nutrients are controlled by multiple genes. The introduction of multiple genes or large fragments of DNA related to these traits from other plants or other organisms into crops and molecular design breeding in accordance with people's wishes is of great significance for improving crop quality and resistance. The introduction of naturally occurring gene clusters or a series of foreign genes that are not linked to one another into the same locus of the plant genome may lead to the emergence of new traits controlled by multiple genes. Not only that, the simultaneous insertion of large fragment gene clusters or gene clusters can also overcome the positional effects and reduce gene silencing and other undesirable phenomena to some extent. At present, the expression vector commonly used in plant transformation is about 7-9 kb in size, and generally can only carry 1 or 2 functional genes. Using such a vector to carry out genetic engineering on the quantitative traits of crops requires multiple repeated transformations of the same genotype. It takes a long time and the conversion and expression effects are not necessarily satisfactory. The BIBAC vector can overcome the shortcomings of commonly used vectors, realize the transformation of multiple genes or large fragments of DNA into plants, and has broad application prospects in crop molecular design breeding. The use of BABIC vectors to transform rice, wheat, corn and other grass crops has not been reported. We have successfully tried BIBAC transformation in rice.
3.7 Trend of New Industrialization of Genetically Modified Crops
The gold rice cultivated by Swiss scientists is rich in vitamin A and is very important for human health and the solution to famine. People are looking forward to the commercial production of gold rice. Chinese scientists have taken the lead in introducing pest-resistant genes, herbicide-resistant genes, and anti-blight blight genes into rice in the world. Currently, they are undergoing environmental release and production trials and are expected to achieve industrial production in recent years. Roundup Ready wheat grown by American scientists showed high resistance to herbicides, showing a promising industrialization prospect. The anti-mV mosaic disease transgenic wheat grown by Chinese scientists is currently undergoing environmental release and production trials. The high gamma-linolenic acid content cultivated by American scientists is high in soybean and high in tryptophan content. The high unsaturated fatty acid content rape cultivated by Canadian scientists is of great value in preventing cardiovascular disease, skin care and improving food nutrition.
In developing countries, the contradiction between population growth and food shortage is increasingly acute. At the same time, accompanied by the reduction of arable land, biological cultivation and the deterioration of living environment, genetically modified technology is the most effective way to solve these problems. In developed countries, the cultivation of genetically modified crops has produced tremendous economic benefits. It is expected that the global trade volume of GM crops will reach 5 billion U.S. dollars in 2005, and will reach 10 billion U.S. dollars in 2010. The need for high-yield, high-quality, disease-resistant, and reversible organisms, and the pursuit of low-cost, high-output, will undoubtedly promote the in-depth study of genetically modified technologies and the large-scale industrialization of genetically modified organisms.
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