Research, Development and Utilization of Transgenic Plants

A transgenic plant refers to a plant that uses a recombinant DNA technology to introduce a cloned good gene of interest into a plant cell or tissue and express it therein to obtain a new trait. This technology overcomes the limitations of sexual hybrids in plants and expands the scope of gene communication indefinitely. It can introduce genes from bacteria, viruses, animals, distant plants, humans, and even humans into plants, so its application prospects are very broad. Since 1983, the world’s first transgenic plant (genetically modified plant, GMP) - tobacco has been produced since the introduction of transgenic plants only 20 years ago, but its research and application has been very rapid development. From 1996 to 2005, the world's transgenic plants were planted in 21 countries and their area increased from 1.7 million hectares in 1996 to 90 million hectares in 2005, an increase of 50 times. The world’s only seed sales of GM crops reached US$5.25 billion in 2005, 63 times more than in 1995. Although the associated safety issues of transgenic plants and public attitudes, technical barriers to trade, and complex factors such as ethics and religion are intertwined into a high-tech political and economic issue, they have become the focus of international and domestic attention and hot spots. However, the brilliant development prospect of transgenic plants is beyond doubt.
1Research progress of transgenic plants
1.1 Types of Target Genes Obtaining useful genes of interest is a basic prerequisite for genetic engineering. In recent years, foreign genes applied to plants have included antiviral, insect-resistance, herbicide-resistance, changes in protein content, male infertility, changes in flower color and flower shape, and extended shelf life. These genes have been transferred to tobacco, respectively. , potatoes, cotton, tomatoes, soybeans, corn, rape, alfalfa, petunia and other plants. So far, more than 100 plant target genes have been isolated and nearly 200 species of transgenic plants have been obtained. Nearly 1,000 transgenic plants have been approved for field trials. More than 50 plant species have been approved, and 48 have been approved. The genetically modified plant species are commercialized and the genes transferred are of the following major types.
1.1.1 Herbicide Resistant Genes This class of plants exhibited resistance to different types of herbicides due to the transfer of herbicide resistance genes. Herbicide-resistant crops such as glufosinate transgenic winter rape, glyphosate resistant crop soybean, corn, cotton, rape, sunflower, sugar beet, anti-sulfonylurea have been obtained so far. Herbicides Genetically modified crops Soybeans, Cotton, Bromide-resistant GM crops Rape, Wheat, Cotton, Tobacco, Atrazine transgenic crops Soybean, Corn n, Antioxazolone herbicides Genetically modified crops Corn, Oilseed rape, Beet, wheat, rice, and dehalogenase transgenic anti-herbicide crops. In addition, the brominated benzonitrile-poisoned BXn gene and the 2,4-D-poisoned tfDA gene were also successfully expressed in herbicide-resistant crop breeding.
1.1.2 Insect resistance genes The scientists of Belgian Plant Genetics Co., Ltd. introduced the Bacillus thunringiensis poisoning protein gene into tobacco for the first time in 1987 and showed resistance to the tobacco budworm larvae. After more than 10 years of development, great progress has been made and large-scale commercial applications have been realized. There are two kinds of insect-resistant genes: one is the Bt insecticidal protein gene, which is from Bacillus thuringiensis. The insecticidal toxicity is the parasporal crystal protein, and it belongs to Lepidoptera, Diptera and Coleoptera. Insects are toxic and have been introduced into cotton, corn, rice, tobacco, tomato, potato, juglans sp., populus sp., larix sp., etc.; the other is protease inhibitor gene , It can inhibit the protease activity, interfere with the digestion of pests and cause its death. It is a self-defense response of plants to insect pests, mainly serine, cysteine, metal containing, aspartame, which has been introduced into cotton and tobacco. , tomato, solanum nigrum and so on. According to the types of genes used for transformation, the development process of the insect-resistant transgenic plants can be roughly divided into two generations: the first generation is mainly the Bt insecticidal crystal protein gene, and many of the transgenic crops it produces are already commercialized. Production, such as tobacco and tomato plants that have acquired the Bt insecticidal crystal protein gene, and the second generation, which has been transformed into a highly efficient insecticidal protein gene outside the Bt insecticidal crystal protein gene, is largely in the laboratory stage for this generation of transgenic crops. A few entered the field trial. The insect-resistant genes have been used most successfully in cotton crops. The Bt genes obtained from transgenic cottons have been reported to include CrylA (b), CrylA (c), CrylIA and CrylVA; the countries involved are the United States, China, Australia, Egypt, France, India, the former Soviet Union, Thailand and so on. The protease inhibitor genes that have been obtained in transformed plants include: soybean trypsin inhibitor gene (SKTI), cowpea trypsin inhibitor gene (CpTI), and mushroom trypsin inhibitor gene (API); among them, CpTI The plant species of the gene are the most numerous, including more than 10 species such as apple, rape, rice, tomato, sunflower, sweet potato, tobacco, and potato. The study of transgenic CpTI cotton in China has been carried out for many years, and it has obtained CpTI gene and Bt+CpTI double gene cotton, and has begun commercial production. In addition, the lectin gene (GNA) was also expressed on at least 10 plants such as rapeseed, tomato, rice, sweet potato, sugar cane, sunflower, tobacco, potato, soybean, and grape, and all exhibited certain insect resistance.
1.1.3 Resistance gene
In 1986, the United States Beachy research team introduced tobacco mosaic virus (TMV) coat protein gene (CP) into tobacco for the first time to cultivate tobacco plants resistant to TMV, which opened a new way for anti-virus breeding. Techniques for increasing the antiviral ability of plants by introducing a coat protein gene of a plant virus have been tested in various plant viruses, such as Liang Xiaoyou et al.'s antiviral CMV-f family gene and insect-resistant B-toxin gene. Tomatoes were introduced and regenerated tomato plants were obtained. The resistance genes that have been introduced at present are: Anti-tobacco Mosaic Virus (MP), Anti-Bacterial Blight, Cotton Fusarium Wilt, TMV and CMV , Wheat resistance to scab, sheath blight, and root rot genes, and resistance to bacterial blight of rice, peanut, tomato bacterial wilt, Chinese cabbage soft rot, citrus canker, mulberry, eucalyptus bacterial wilt, Root club disease and other studies. Plants that have acquired transgenic disease resistance traits include: tobacco, tomato, cotton, barley, oatgrass, wheat, potato, rice, and the like. In addition to the coat protein gene as an effective pathway, in recent years, domestic and foreign laboratories are exploring new methods for antiviral gene engineering, including satellite RNA, replicase genes, and viral replication inhibitory factors, ribosome inactivating proteins, and pathogenic-related diseases. Proteins, nucleases, etc. Bacterial disease and fungal disease resistance genetic engineering research is basically still in the laboratory stage. Transgenic cucumber-resistant mosaic pepper sweet peppers and tomatoes cultivated in China have achieved commercial production.
1.1.4 Antiadversity Genes A number of genes related to stress metabolism have been isolated, including the proline synthase gene, antifreeze protein (AFP) gene, and Arabidopsis chloroplast 3 Phosphoglycerol acyltransferase gene, the honeysuckle sugar synthase gene associated with drought resistance, and some plant desaturase genes. Research on the isolation, cloning and transformation of stress-resistant genes in China has achieved certain progress. Salt- and alkali-resistant genes have been cloned. Medicinago sativa resistant to 1% NaCl has been obtained through genetic transformation. The NaCl strawberry, the tobacco resistant to 2% NaCl, and the reversible genetic engineering crop have entered the field experiment stage. Liu Yan and others obtained transgenic maize plants with significantly increased salt tolerance. Zhang Wei and others obtained genetically modified tomatoes with improved salt tolerance.
The Department of Biological Engineering of Shihezi University in Xinjiang used the pollen channel method to introduce the salt-tolerant genes of reed and bacteria into wheat, and developed new lines of anti-saline-alkaline transgenic wheat 253, 282, 221 etc. The salt-tolerance capacity was significantly enhanced. Sakamoto et al. obtained two transgenic rice plants with increased salt tolerance and cold tolerance. Capell et al. used the CaMV35S promoter to overexpress Ade in rice, inhibited the degradation of chlorophyll under drought stress, and improved drought resistance. Steponkus et al. found that the cold tolerance of chloroplasts and protoplasts in Arabidopsis transferred to Corl5a increased. Mekersie et al. transferred the eDNA of the Mn-SOD cloned from tobacco into the pods under the 35S promoter. The transgenic larvae were compared in two winter field trials, and the survival rate in winter was greater than that in non-transplanted plants, with an average increase of 25%. The United States has used resistance genetic engineering technology to develop herbicide-tolerant soybeans and freeze-resistant strawberries, which have been used in field production. Stanford University in the United States introduced cactus genes into wheat, soybean, and other crops to develop new drought-resistant and new varieties.
1.1.5 Improved quality Genetic quality improvement mainly involves the content of proteins, the composition of amino acids, the composition of starch and other polysaccharide compounds, and lipid compounds. Methionine-rich transgenic tobaccos, transgenic rice with reduced amylose content, and genetically modified rapeseed with a lauric acid content as high as 40% have all succeeded, and some have entered field trials. In addition, mature transgenic tomatoes and altered floral genetically modified roses have also been commercialized. “The story of Kimmi” (in which two genes of daffodils and one gene of a bacterium were introduced into a rice called T309 to obtain a new rice variety. The new rice thus obtained was rich in iron, Zinc and carotene, which can be converted to vitamin A, can prevent anemia and vitamin A deficiency, and the rice is golden brown. It tells us that genetically modified technology has improved the quality of rice and it has become possible to solve human malnutrition. In our country, the Zein gene was introduced into potato, and the content of essential amino acids in the tubers of field transgenic plants increased by more than 10%, while the increase in sulphur-containing amino acids was particularly significant. In addition, the methionine-rich transgenic tobacco Transgenic rapeseed with reduced amylose content has been successively successful, and some have entered field trials. Extension of mature transgenic tomatoes and change of color of transgenic roses have also been commercialized.
In addition, anti-premature aging genes that use transgenic technology to inhibit ethylene synthesis or promote cytokinin synthesis have also been reported. Transgene production in bioreactors can be used to produce oral vaccines, industrial enzymes, fatty acids, genetic drugs, and the like. The more successful example is the use of genetically modified rapeseed to produce non-edible industrial oils, including dodecyl lauric acid, which is used as a detergent for soaps, and genetically modified rapeseed is also used as a raw material for the production of lubricating oils and nylon. 6 octadecenoic acid produced by margarine. In addition, transgenic plants are also used to produce high-molecular materials such as biodegradable polyhydroxybutyl plastics and natural cotton and polyester blends. Among all the genes of interest, such as insect resistance, disease resistance, herbicide-resistance, and the like, which are inputs for reducing input, are often referred to as first-generation transgenic plant traits; and second-generation transgenic traits are referred to as increasing yield traits, such as quality. Improvements, value-added processing, specialty products, medicine, and health care are more acceptable to consumers.
1.2 Methods for the transformation of target genes Since the production of transgenic plants, methods for the genetic transformation of various genes have emerged. Up to now, several sets of plant genetic transformation systems have been established. They have different characteristics and are used in different receptor plants. The correct selection of the transformation system has also become a prerequisite for the successful transformation of a plant gene.
The genetic transformation of a target gene refers to the process of introducing foreign DNA into a plant cell through a vector, a medium or other physical and chemical methods and obtaining integration and expression. The way to achieve this transformation is called conversion system. The system in which foreign DNA mediates its transformation by vectorization is called a gene vector transformation system. At present, more than ten genetic transformation methods have been established. According to the principle of transformation system, they can be divided into three types of transformation systems:
(1) A transformation system using a plasmid DNA or the like as a vector, such as the Agrobacterium method. About 80% of the transgenic plants obtained so far were transformed with Agrobacterium tumefaciens.
(2) Direct transformation of foreign genes into human recipient cells via physical and chemical methods without any carrier, such as microneedle injection and particle bombardment.
(3) Using the plant's own reproductive system germplasm cells, such as pollen grains or other cells, as the media's transformation system, such as the pollen tube channel method.
The characteristics of commonly used plant gene transformation methods are listed in Table 1.
2 Application of transgenic plants
2.1 Research of transgenic plants Development history The world's first transgenic plant - tobacco was introduced in the United States in 1983. In 1986, the first transgenic plants, insect-resistant and herbicide-resistant cotton, were field tested. It is worth noting that China became the first commercialized country in the world in 1992, and it pioneered the commercialization of transgenic plants. At that time, it was planted with a pair of transgenic cucumbers resistant to cucumber mosaic virus and tobacco mosaic virus. . In 1993, the first transgenic plant, the ripened tomato, was approved by the US Department of Agriculture for commercial production. In 1994, the first transgenic plant product, Flavr Savr, was approved by the US Food and Drug Administration (FDA) to enter the market. Since 1983, the research and development of plant genetic engineering has progressed rapidly in less than 20 years.
2.2 Global application of transgenic plants From a global perspective, the successful application of the commercialization of transgenic plants focused on genetically modified crops. Its planted area and sales revenue increased at multiples and developed rapidly. Its overview is as follows:
(1) Planting area. In 1996, the total area of ​​GM crops planted in the world was 1.7 million hectares. After that, there was a substantial increase every year. It was 11 million hectares in 1997, 27.8 million hectares in 1998, 39.9 million hectares in 1999, 44.2 million hectares in 2000, and 52.6 million hectares and 58.70 million hectares in 2001 and 2002 respectively. By 2005, the area of ​​global genetically modified crops had been planted. It reached 90 million hectares, an increase of 11% from 81.1 million hectares in 2004, 53 times that of 1996.
(2) Planting status of 4 major GM crops. In 2005, the four largest genetically modified crops were soybean (5.44 million hectares, accounting for 60% of the area of ​​similar crops), cotton (9.8 million hectares, accounting for 28% of the area of ​​similar crops), and rapeseed (4.6 million hectares). 18% of the area of ​​similar crops, corn (21.2 million hectares, 14% of the area of ​​similar crops).
(3) Planting distribution. By 2005, 8.5 million rural households in 21 countries had planted genetically modified crops. The United States, Argentina, Brazil, Canada, and China are still the world's major growers of GM crops. About 20% of the 49.8 million hectares of genetically modified crops grown in the United States (55% of the world's planted area) are mixed genetic products containing 2 or 3 genes. In 2005, the first three-gene product in the United States was corn. Hybrid genetic products are an important development trend in the future. The United States, Canada, Australia, Mexico and South Africa have already begun to grow such crops, and the Philippine government has also approved the cultivation. The fastest-growing country in 2005 was Brazil, with an initial forecast of an increase of 4.4 million hectares (5 million hectares in 2004 and 9.4 million hectares in 2005); India’s annual growth rate is the fastest, almost three times growth. From 500,000 hectares in 2004 to 1.3 million hectares in 2005. In 2005, 21 countries that cultivated biotech crops included 11 developing countries and 10 industrialized countries. According to the order of planting area, they are the United States, Argentina, Brazil, Canada, China, Paraguay, India, South Africa, Uruguay, Australia, Mexico, Romania, Philippines, Spain, Colombia, Iran, Honduras, Portugal, Germany, France and the Czech Republic.
(4) Sales of transgenic crop seeds. In 1995, the global sales of genetically modified crop seeds (including some technical fees) were US$84 million. From 1996 to 2003, they were 347 million, 1.113 billion, 2.259 billion, 2.931 billion, 3.045 billion, 4.25 billion, and 4.75 billion U.S. dollars. In 2005 it increased to US$5.25 billion and sales increased by 63 times in 10 years, which is equivalent to 15% of the global crop protection market in 2005 (US$34.02 billion) and 18% of the global commercial seed market (US$30 billion). The $5.25 billion biotech crop market includes 2.42 billion U.S. dollars of Bt soybeans (46% of the global market for biotech crops), 1.91 billion U.S. dollars of Bt corn (36%), and 720 million U.S. dollars of Bt cotton (14%). 210 million U.S. dollars of Bt canola (4%). According to the forecast of the International Agency for the Application of Agricultural Biotechnology (ISAAA), it is expected to exceed US$5.5 billion in 2006 and reach US$20 billion in 2010.
(5) traits of transgenic crops. At present, the traits of genetically modified crops are mainly concentrated on herbicide resistance and insect resistance. Herbicide-tolerant herbicides have been the most important feature for the first decade from 1996 to 2005, followed by insect resistance and mixed genes. In 2005, the global planting area of ​​genetically modified crops reached 90 million hectares, of which 71% (63.7 million hectares) of herbicide-tolerant soybeans, corn, and rapeseeds, 18% (16.20 million hectares) of Bt crops, and ll% of mixed genetic crops ( 10.10 million hectares). Mixed genetic crops were the fastest growing between 2004 and 2005, with a growth rate of 49%, herbicide-tolerant growth rate of 9%, and pest growth rate of 4%.
2.3 Research and application of transgenic plants in China Research on transgenic plants in China began in the 1980s. The 863 program launched in 1986 has played a key role in guiding, leading, and radiating. From 1997 to December 2003, the Ministry of Agriculture of the People's Republic of China approved 243 environmental assessment applications, 108 production tests, and 79 production safety certificates. The transgenic plants that have entered the environmental release phase include: rice, corn, wheat, potato, Hetao melon, papaya, soybean, rapeseed, poplar, tobacco, tall fescue, ryegrass; the transgenic crops that have been commercialized are: Cotton (46 items), chili (1 item), sweet pepper (4 items), petunia (1 item), tomato (6 items), total 58 items.
In the same period, the United States, Canada and the European Union had a total of 14,485 environmental releases and 118 commercialized permits. The United States has the largest number of genetically modified organisms, with 106 ports. In terms of application, the research and development of transgenic insect-resistant cotton in China has been rapidly developed. It is the second country with independent intellectual property rights after the United States and is the first successful example of domestic genetic engineering for agricultural production. From 1998 to 2003, the cumulative planting of over 8 million hectares in the past six years has increased, and the proportion of total domestic cotton planting area has increased year by year. In 2002, the area of ​​Bt cotton planted reached 2.1 million hectares, accounting for 51% of the total area of ​​4.1 million hectares of cotton, and exceeded 50% for the first time. The application of transgenic insect-resistant cotton can reduce the use of pesticides by 40% to 70%, greatly reducing pesticide poisoning accidents and generating huge social, economic and ecological benefits. At the same time, it promoted the research and development of biotech products such as insect-resistant transgenic rice, maize, and poplar. China's genetic industry is quietly forming.
Undoubtedly, with the further rationalization and refinement of plant transgenic technology, a new generation of transgenic plants will be safer and more environmentally friendly to humans. This will fundamentally resolve people’s concerns and the fierce disputes over safety, as a result of transgenic technology. The further development will create a good social environment so as to create a better human future.