Transgenic technology

 

 

Transgenic technology allows for the development of plants with unique qualities in a much shorter time than traditional plant breeding, as well as the introduction of characteristics that cannot be accomplished by plant breeding alone. The production of genetically modified plants has been fuelled by rapid advances in gene editing and the desire to improve agricultural productivity, both to reduce pesticide and fertiliser inputs and to improve quality. However, assessing the risks of transgenic plants to human health and the environment is a major roadblock to public acceptance and commercialization. Regulatory agencies and consumers are worried about the environmental protection of genetically modified organisms (GMOs), and demand that commercial transgenic plants be free of unwanted genes like antibiotic resistance marker genes and vector backbone sequences. Recent research has clarified these issues and outlined a path to their resolution. We divide the methods for developing genetically modified plants into two categories: (A) plant transformation technologies and (B) techniques for removing selectable markers.

 






Transgenic technology.

Agrobacterium is the most popular method for plant genetic engineering because of its wide host range. It has been used to transform a variety of crops, medicinal plants, and aromatic plants. In the early 1980s, the main findings that led to the use of the Agrobacterium Ti plasmid as a vector for plant transformation and kanamycin resistance genes for transformed cell selection were released. Numerous genetic-transformation protocols for various plant species have been developed during more than three decades of Agrobacterium biotechnology research, and a wide range of Agrobacterium-related patents have been asserted. Agrobacterium-mediated transformation is a single-cell transformation mechanism that prevents mosaic plants from forming. This soil bacterium has the natural ability to transform its host by delivering a well-defined DNA fragment from its tumor-inducing (Ti) plasmid, called the transferred (T) DNA, into the host cell. T-DNA penetration into the host genome and expression of its encoded-native bacterial genes results in neoplastic growth and tumour formation on infected plants, indicating a successful genetic-transformation event. The molecular basis for almost all Agrobacterium-mediated genetic transformation protocols is replacing the bacterial genes with different genes of interest, which has no effect on the transformation process. Although this is a highly successful transformation process, some plants react better to it than others. The ability of A. tumefaciens to infect the cells and insert its T-DNA into the plant genome until it is killed, as well as the ability of the transformed cells to be cultured to form a whole plant, are both needed for a successful transformation. Efficient Agrobacterium-mediated gene transfer methodologies have been developed primarily for dicotyledoneous plants such as those in the Solanaceae family. (tobacco, tomato and potato) that have given the best results. At the negative end of the scale are the monocotyledons, including the four species of wheat, rice and maize in which A. tumefaciens does not readily infect. Since the Agrobacterium transformation process involves many stages, different pre- and post-agroinfection strategies have been employed by each worker to achieve maximum transformation efficiency.In addition to these strategies; there are reports of using surfactants during inoculation and co-cultivation to increase T-DNA delivery. Increasing concentrations of Silwet L-77 up to 0.04% had positive effects on T-DNA delivery as measured by the number of immature embryos with GUS foci and the number of GUS foci per embryo. However, concentrations higher than 0.05% reduced survival and callus formation in freshly isolated immature embryos and an optimal concentration of 0.01% was chosen. Positive effects of surfactants were also reported in study which used Silwet and pluronic acid F68 at 0.02%. Silwet has been used at concentrations as high as 0.05% for pre-cultured embryos and calli.

Sonication assisted agrobacterium-mediated transformation (SAAT). This method uses sonication to create thousands of micro wounds on and beneath the surface of plant tissue, allowing Agrobacterium to permeate the tissue. Using mature embryos as explants, it was used to research transient GUS expression in cotton. Since ultrasound promotes the uptake of plasmid DNA into the cells of flax hypocotyls and cotyledons, the SAAT cocultivation technique could be a promising method for improving transformation efficiency in flax. The length of the procedure and the frequency with which it is used must be optimised in order to improve transition performance.

Agroinfiltration is a term that refers to the process of filtering Agrobacterium tumefaciens transient transformation assay (agroinfiltration) is a quick, rapid, and repeatable plant transient gene expression technique. It's been used to investigate foreign gene expression, hypersensitive reactions, gene silencing, promoter function, and the discovery of new disease-resistance genes. In this step, the desired gene is first inserted into an Agrobacterium strain. After that, the suspension is inserted in a syringe (without a needle) and injected into the airspaces within the leaf via stomata by pressing the tip of the syringe against the underside of a leaf while applying gentle counter-pressure to the opposite side of the leaf. The Agrobacterium transforms the gene of interest to a portion of the plant cells and then transiently expresses it.Bechtold et al. used vacuum infiltration for the first time in Arabidopsis transformation, which was later improved for efficiency. Recently, this protocol was further modified by excluding vacuum infiltration and spraying plants with bacterial spores instead. pBin and pB, which was generated by adding a marker gene to pBin, are two of the most common binary vectors. pPZP vectors and pCAMBIA vectors are two other common vector series (www.cambia.org). T-DNA and the vector backbone make up a binary vector. T-DNA is the portion of DNA that is delimited by the right (RB) and left (LB) boundary sequences and may contain several cloning sites, a plant selectable marker gene, a reporter gene, and other genes of interest. For E. coli, the vector backbone contains plasmid replication functions. A. coli and E. coli tumefaciens, selectable marker genes for the bacteria, and plasmid mobilisation mechanism between the bacteria and other accessory components, if desired. In binary vectors, several finer changes have been made. The series of pBIN19 has been completed. A new series of pPZP vectors has been developed that are small in size and stable in Agrobacterium. Its improved version, pBIN, has several additional single restriction sites in the multiple cloning sites (MCS). The pCAMBIA series of vectors were built using the pPZP vector backbone, with nptII, hpt, or bar as selection markers and gus or gfp as reporters. For rice transformation, the pCAMBIA vectors are commonly used. Topfer et alpRT100 .'s series plant expression vectors allow for the development of gene cassettes containing the CaMV 35S promoter and its polyA signal. These cassettes can be removed and inserted into binary vectors' MCS. A recent survey of higher plant transformations mediated by A. tumefaciens showed that pBin19 derivatives were used in 40% of the studies and pPZP vector derivatives in 30% of them.acterial suspension produced the best results.

Method of using a biolistic gun. Microparticle bombardment (Biolistic gun) is a highly effective method for transferring genes to plant cells that has been widely used to transform plants that are resistant to Agrobacterium transformation. Cereals and legumes, which are the world's most significant food crops, are included. The technology involves the use of DNA-coated gold or tungsten particles that are pushed at high speeds into the plant tissue that needs to be transformed. Some particles break through the cell walls of plants and introduce foreign DNA into healthy cells. Christou et al. were the first to record stable transition events as a result of microprojectile bombardment shortly after. They were able to deliver viable DNA into immature soybean embryos and generate stable transformed callus material from isolated protoplasts using an electrical discharge gun. Agrobacterium or particle bombardment for gene transfer?

There has been a lot of discussion about whether particle bombardment or Agrobacterium is the best way for producing genetically modified plants. Bombardment technologies were designed to overcome the incompatibility of many plant species' tissues with the Agrobacterium vector. Particle bombardment, as a physical method, can be applied to a wide range of biological systems and can be used for both transient expression studies (e.g., promoter analysis) and the development of stable transformants.68,69 Particle bombardment has also been used to wound plants in order to promote Agrobacterium transformation.70 Another criticism of this approach is that there is a high risk of DNA being introduced into a functioning gene.

Electroporation is a term that refers to the process of transferring Electroporation is a mechanical technique for introducing polar molecules across the cell membrane into a host cell. A strong electric pulse disrupts the phospholipid bilayer, allowing molecules like DNA to move into the cell.97 Electroporation typically requires 10,000–100,000 V/cm (varying with cell size) in a pulse lasting a few microseconds to a millisecond. The phospholipid bilayer of the membrane is disrupted by the electric pulse, resulting in the creation of temporary aqueous pores. The electric potential across the cell's membrane increases by around 0.5–1.0 V at the same time, allowing charged molecules to pass through the pores. Electroporation allows DNA, RNA, proteins, drugs, and dyes to be introduced into recipient cells through induced electropores. The first reports of DNA delivery into plant protoplasts9were made using PEG-mediated protoplast transformation, and Paszkowski et al. developed the first transgenic plants using this method with tobacco.

Liposomes are involved. When phospholipids are hydrated, they form microscopic spherical vesicles called liposomes. Most commercial liposomes are made up of phospholipids, cholesterol, a variety of lipids, and sometimes polymers. They may contain a wide range of molecules, including DNA. These are used for protoplasts, membrane fusion-mediated transfection (lipofection), and endocytosis. Liposome-mediated transformation is time-consuming and ineffective. Few reports on liposome-mediated transformation and transgenic plant regeneration have been published in plants, e.g. tobacco, and wheat One of the drawbacks of liposomes made from natural phospholipids is their low stability in biological environments, where they are quickly cleared. Recently, the efficiency of bioactive-beads-mediated plant transformation has been enhanced by using a DNA-lipofectin complex as the entrapped genetic material instead of naked DNA as in the traditional process.

Microinjection is a technique for injecting a small amount of this approach involves injecting DNA into the nucleus or cytoplasm with a glass micro capillary-injection pipette using a micromanipulator. Despite its widespread use for the transformation of large animal cells such as frog egg cells or mammalian embryonic cells, its usefulness for plant transformation is limited due to a number of drawbacks. First, the plant cell wall acts as a barrier to glass micro tools; second, microinjection of protoplasts causes the release of hydrolases and other toxic compounds from the vacuole to the cytoplasm, resulting in rapid protoplast death; and third, the removal of vacuoles prior to microinjection, although having no effect on protoplast viability, causes a significant reduction in the capability for morphological analysis. Furthermore, the process is time-consuming and necessitates the use of a costly micromanipulator. Despite the success of the technique in incorporating plasmids and chromosomes into plant cells, only a small number of transgenic plants were found in tobacco, petunia, rape, and barley.

Electrophoresis is a technique for separating proteins. Despite Ahokas, Griesbach, and Hammond's claims of simplicity and low cost, electrophoresis as a plant transformation tool is of negligible value. The embryos (explants) inserted between the tips of two pipettes connected to electrodes were not viable, which was the key reason for its failure. Despite the fact that Ahokas' first attempts yielded plants from barley embryos, none of them expressed the uidA gene borne by the plasmid used for transformation. The only active transgenic plants to date have been obtained for the Calnthe orchid.

 

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