Manipulating genetically modified plants so that they can grow rapidly without being restricted by natural factors and survive in extreme environments is a complex and challenging process that involves multiple steps and techniques.： ### Gene selection and Cloning 1. **Identify the target gene** -**Growth-related genes**: For example, select genes that can promote cell division and elongation. Genes related to cytokinin synthesis can increase the frequency of plant cell division, thereby speeding up plant growth. Efficiently expressed such genes were selected from some fast-growing plant varieties, such as the key genes for cytokinin synthesis in some tropical fast-growing plants. -**Resistance-related genes**: According to different extreme environments, select corresponding resistance genes. In an arid environment, the gene encoding proline synthesase can be selected. Proline can help plant cells maintain moisture and enhance drought resistance. Such genes are usually found in some drought-tolerant wild plants, such as desert plants. 2. **Gene cloning** - Using molecular biology techniques, DNA fragments of the target gene are extracted from the donor organism (such as the plant varieties mentioned above). Gene amplification is carried out by PCR (polymerase chain reaction) technology to obtain a sufficient amount of target gene. For example, design specific primers, use plant genomic DNA as a template, and optimize the PCR reaction conditions, such as suitable temperature cycles, primer concentrations, etc., to amplify a large number of target genes. ### Carrier construction 1. **Choose the right carrier** - Commonly used vectors are plasmids, viral vectors, etc. For plant genetic engineering, Ti plasmid (a plasmid in Agrobacterium rhizosum) is a commonly used carrier. It can integrate exogenous genes into the plant genome. Select a Ti plasmid vector with a suitable polyclonal site (MCS) in order to insert the target gene. 2. **Construct recombinant vector** -Insert the cloned target gene into the polyclonal site of the vector. Restriction endonucleases are used to cleave the vector and target gene so that they produce complementary sticky or flat ends, and then the two are connected by DNA ligases. For example, restriction endonucleases such as EcoRI and HindIII are used to cleave the vector and target gene respectively, and then T4 DNA ligase is used to connect them into recombinant vectors to ensure the correct insertion direction and code reading frame of the gene. ### Transforming plant cells 1. **Agrobacterium-mediated transformation method** -For most dicotyledonous plants, Agrobacterium-mediated transformation is commonly used. The recombinant vector is introduced into Agrobacterium cells, and then plant implants (such as leaves, stems, etc.) are infected with a bacterial solution containing recombinant agrobacterium. Agrobacterium transfers and integrates T-DNA (transfer DNA) from recombinant vectors into the plant cell genome. For example, the transformed agrobacterium is inoculated on a medium containing plant implants, and cultured for a period of time under suitable conditions (such as specific temperature, light, etc.) to promote the interaction between agrobacterium and plant cells. 2. **Gene gun-mediated transformation method** -For plants where agrobacterium-mediated transformation is more difficult, such as monocotyledonous plants, gene guns are commonly used to mediate transformation. The gold powder or tungsten powder particles wrapped in recombinant vectors are injected into plant cells at high speed with a gene gun. Adjust the parameters of the gene gun, such as pressure, particle size, etc., so that the vector can effectively enter the cell and integrate into the genome. For example, an appropriate amount of recombinant vector is mixed with gold powder, loaded into the sample-carrying chamber of the gene gun, and plant cells are bombarded. ### Screening and identification 1. **Screening of transformed cells** -Cultivate transformed plant cells on a medium containing selected markers. For example, if the vector carries an antibiotic resistance gene (such as kanamycin resistance gene), then kanamycin is added to the medium. Only cells that have successfully integrated the recombinant vector can grow on the medium containing kanamycin, thereby screening out transformed cells. 2. **Identification of transgenic plants** -**PCR identification**: Extract the genomic DNA of transgenic plants and use it as a template for PCR amplification to detect the presence of the target gene. If a fragment that matches the size of the target gene can be amplified, it is preliminarily proved that the target gene has been integrated into the plant genome. -**Southern hybridization identification**: Further verify the situation of gene integration. The genomic DNA is enzymatically cleaved, electrophoresis is performed, and then hybridized with the labeled target gene probe. If a specific hybridization band appears, it means that the target gene has been integrated into the plant genome, and information such as the number of copies of the integration can be determined. -**Northern hybridization or RT-PCR identification**: Detect the transcription level of the target gene to determine whether it is normally transcribed into mRNA in transgenic plants. -**Western hybridization identification**: Detect the expression of the protein encoded by the target gene and verify whether the transgenic plants can correctly express the target protein. ### Optimization and regulation 1. **Gene expression regulation** -The expression level of the target gene can be regulated by adding different promoters in front of it. Constitutive promoter such as CaMV 35S promoter can make genes in various tissues of plants and