1. Introduction

Transgenic plants modify their genomes through genetic engineering by adding foreign genes or removing harmful genes. The alien genes inserted into plants can be different species, or even kingdoms. The first genetically modified plant was developed by inserting the nptII bacterial antibiotic resistance gene into tobacco. Since then, with the rapid development of plant molecular biology and genetic engineering technology, a variety of important agronomic characteristics such as pest resistance and drought resistance have been formed, from binary plants to genetically modified monomers. The main purpose of GM plant production is to produce crops with ideal characteristics, quality and high yield. In addition to being beneficial to the agricultural sector, these plants can also be used as factories for the production of pharmaceutical proteins.

2. Application of transgenic plants

2.1. Resistance to biotic or abiotic stresses

Stress occurs naturally due to pressure from other organisms in the same ecosystem. These include bacteria, viruses, herbivores, or native plants.

2.2. Improving crop yield and nutritional value

Malnutrition is a major health problem, particularly in underdeveloped and developing countries, because of limited access to nutritious food. Genetic engineering of major crops has become one of the more effective ways to address this problem.

2.3. Transgenic plants as bioreactors for recombinant proteins

Recombinant proteins produced using mammalian expression systems produce low yields and are expensive, while bacterial systems cannot be modified after conversion in complex protein formation. As a result, production methods have shifted to plant cell systems, providing a cheaper and better alternative source for the production of recombinant proteins. Recombinant proteins produced by genetically modified plants include antibodies, metabolites or metabolites, proteins, and vaccines

3. Gene constructs

A simple functional gene construct consists of a promoter region, gene coding region, and terminator/stop region. Additionally, certain gene constructs may contain special sequences such as an enhancer, silencer, or reporter sequences depending on the nature of study. Plant transformation always starts with the transgene construction. Transgene construct generally has similar elements other than the inclusion of the gene of interest and selectable markers. Proper genetic structure is critical to the successful production of the ideal GM line.

（1）A typical plant gene

（2）Promoters/enhancers

Table 1 shows selected promoters used in plant transformation.

Promoter	Source	Activity
CaMV35S	Cauliflower mosaic virus	Constitutive
Ubiquitin RUBQ1, RUBQ2 and rubi3	Rice	Constitutive
Ubiquitin Gmubi3	Soybean	Constitutive
SCR, SRK	Brassica rapa	Pollen and stigma specific
Exo70C2	Arabidopsis	Pollen and root specific
LMW Glu, HMW Glu-1D1	Wheat	Seed specific
Expansin PcExp2	Sour cherry	Ripened fruits
Potato class I patatin	Potato	Tuber/storage organ specific
NtHSP3A	Tobacco	Stress inducible

Table 1.

Examples of promoter used in plant transformation.

Source: Adapted from Hernandez-Garcia et al.

Enhancers are short areas of the gene (50 to 1500 bp) that can be identified and bound by activated proteins. These proteins, also known as transcription factors, bind to enhancers to form enhancers that bind to transcription factor complexes that will later interact with the intermediate complex (TFIID), ultimately helping to recruit RNA polymerase II.

3.3. Reporter genes

Reporter genes are genes attached to the regulatory sequences or to gene of interest to allow for detection of the transgene expression as well as the localization of expressed proteins. Reporter gene sequences encode proteins or products of the protein after being catalyzed for detection through instruments or simple assays. In contrast, selectable marker genes such as antibiotic genes, herbicidal-resistant genes, and anti-metabolic genes confer resistance toward certain chemical agents, which inhibit nontransgenic plant development.

3.4. Problem posed by antibiotic resistance reporter genes

The plant conversion techniques currently available are quite effective, but not perfect. No technology can provide 100% conversion efficiency. In order to distinguish between transformed and unconverted plant cells, markers are required. Antibiotic or herbicide resistance genes are the main selective markers for the selection of conversion agents, which can effectively eliminate non-conversion agents. The effectiveness of an antibiotic resistance system depends on three criteria:

(1) selective agent used should completely inhibit the growth of nontransformed cells,

(2) resistance gene is expressed in transformed cells，

(3) explant used for transformation. 

Table 2 shows some of the antibiotics used in transgenic plant screening.

Table 2.

Selective antibiotics used for transgenic plants screening.

4. Vectors for the production of transgenic plants

A vector acts as a vehicle that transports the gene of interest into a target cell for replication and expression. Common vector is made up of three components: an origin of replication, multicloning site or recombination site, and selectable marker.

4.1. Plasmid vectors

4.1.1. Ti plasmid

The Ti plasmid is large and would become larger with the genes of interest and selectable markers. Large-sized plasmids are cumbersome to handle and have low copy numbers in nature. However, this drawback eventually led to the development of a co-integrative system in combination with the binary vector system which solved the problem for large-sized plasmids.

4.1.2. The co-integrative vector

This co-integrative vector will later be introduced back into the Agrobacterium for transgenic plant transformation. However, the enormous size of the plasmid as a result from the recombination may prove an ominous challenge to be manipulated. Thus, the use of this vector had been discontinued since the binary vector system was introduced.

4.1.3. The binary vector

Viruses are intracellular forced parasites that require molecular mechanisms from specific hosts to replicate. No viruses have been found to infect plants through vectors such as beetles, insects, nematodes and fungi. These viruses have been modified and used as an alternative source for plant conversion; common plant viruses used in GM plant production include broccoli mosaic virus (CaMV), tobacco mosaic virus (TMV), alpha mosaic virus (AMV), potato virus X (PVX) and cow pea mosaic virus (CPMV). Wild plant virus vectors have been improved and adapted to their use with agricultural bacteria as well as plant hosts to improve efficiency levels through two methods. The first method is to design a vector similar to a wild virus that carries genes that can infect plants. The second approach is to develop a "deconstructed" virus that is produced by removing unwanted viral genes, such as coated protein expression genes, and replacing them with functional genes such as the reporter's gene or antibiotics. Resistance genes to promote genetically modified screening.

When researchers found that T-DNA was running independently, a double-grain system called a binary carrier system was developed that did not need to be attached to Ti plasmids. The binary system involves two plasmids, the help vector and the micro vector. Microcarriers are smaller plasmids consisting of T-DNA, and the origin of E. coli and E. coli replication, which allows plasmids to be cloned in E. coli and E. coli. Helper vectors are wild Ti plasmids that do not have a T-DNA area. Wild Ti plasmids are also known as help plasmids because they provide a template for all the genes needed for gene transfer and integration. Together, these two helpers and microcarriers are introduced into agricultural bacteria, which are converted to be used for plant transformation.

4.2. Plant virus vectors

Viruses are intracellular forced parasites that require molecular mechanisms from specific hosts to replicate. No viruses have been found to infect plants through vectors such as beetles, insects, nematodes and fungi. These viruses have been modified and used as an alternative source for plant conversion; common plant viruses used in GM plant production include broccoli mosaic virus (CaMV), tobacco mosaic virus (TMV), alpha mosaic virus (AMV), potato virus X (PVX) and cow pea mosaic virus (CPMV). Wild plant virus vectors have been improved and adapted to their use with agricultural bacteria as well as plant hosts to improve efficiency levels through two methods. The first method is to design a vector similar to a wild virus that carries genes that can infect plants. The second approach is to develop a "deconstructed" virus that is produced by removing unwanted viral genes, such as coated protein expression genes, and replacing them with functional genes such as the reporter's gene or antibiotics. Resistance genes to promote genetically modified screening.