AGROBACTERIUM-MEDIATED TRANSFORMATION: CHALLENGES AND PROGRESS IN PLANT GENETIC ENGINEERING

Introduction

The use of Agrobacterium tumefaciens's inherent capacity for gene transfer to introduce desired genes into plants is a key component of the plant genetic engineering process known as "Agrobacterium-mediated transformation." This strategy is preferred in plant biotechnology since it has been instrumental in improving a number of crop species by transferring important agronomic features. While the approach has several benefits, like reliable transgenic integration and cost-effectiveness, it also has drawbacks, such as low transformation efficiency, problems with specific plant species, and transgene integration complexity.

Let's discuss the creation of transgenic crops by this technique, the particular crops that have undergone modification, and the dangers involved in raising transgenic plants. It also looks at the difficulties that arise from Agrobacterium-mediated transformation and how cutting-edge biotechnology technologies like CRISPR/Cas9 can help to solve these problems, eventually raising the success rate of genetic transformation in corn. By offering useful insights to researchers working on crop genome editing using Agrobacterium-mediated transformation, this review hopes to pave the way for further developments in plant genetic engineering.

The Need for Genetic Engineering in Modern Agriculture

Over the years, conventional plant breeding has unquestionably increased crop output. But as the world's population gets closer to 10 billion, these conventional methods are becoming less and less able to supply the world's increasing demand for food, which is expected to increase by 50% by the middle of the twenty-first century. Additionally, the necessity to produce crops that can thrive in inadequate settings and resist biotic and abiotic challenges is made worse by the urbanization-related rapid loss of cultivable land. To address these issues, genetic engineering (GE) provides a workable and effective answer. Genetic engineering (GE) greatly speeds up the improvement of crop types by precisely modifying a plant's genetic material to incorporate desired qualities through the use of recombinant DNA technology.

Process of Developing Transgenic Plants

There are several important processes in the formation of transgenic plants. These consist of determining and isolating target genes, building expression cassettes, cloning into appropriate vectors, and deciding on the best means of delivering genes. Transgene integration and expression detection requires a robust tissue culture system, selection markers, and biochemical techniques. The International Service for the Acquisition of Agri-biotech Applications (ISAAA) estimates that 190 million hectares worldwide were planted with genetically modified crops in 2019, demonstrating the enormous economic effect of these commodities.

To improve crop types, GE can also upregulate, downregulate, or silence endogenous genes in addition to transgenic expression. Plant transformation has developed into an effective tool for functional genomics research and gene cloning. Techniques for genetic transformation have been developing and can be broadly divided into two categories: indirect and direct approaches. Agrobacterium and virus-mediated gene transfer are examples of indirect methods, whereas sonication, electroporation, microinjection, microprojectile bombardment, and other techniques are examples of direct methods. The two that best exemplify their respective categories are microprojectile bombardment and Agrobacterium-mediated transformation. The biology of Agrobacterium-mediated transformation, the variables influencing its effectiveness, and current developments in the field are the main topics of this paper.

Mechanism of Agrobacterium-Mediated Transformation

A gram-negative bacterium called *Agrobacterium tumefaciens* is frequently found in soil. It naturally infects the injured sections of dicotyledonous plants, causing crown gall tumors. Transfer (T)-DNA is present in the tumor-inducing (Ti) plasmid of *Agrobacterium*, and it integrates into the plant genome to cause tumors. Oncogenes that generate mitogens, such as auxin and cytokinin, which promote unchecked plant tissue growth, are present in the T-DNA area. In genetic engineering, the target gene integrates into the plant genome by replacing the oncogenes in the T-DNA with the gene of interest (GOI).

Application and Constraints of Agrobacterium-Mediated Transformation

You can use agrobacterium-mediated transformation on a variety of explants, such as shoot apices and leaf disks. Horsch and associates developed the leaf disk inoculation technique, which made it possible to introduce foreign genes into a variety of dicotyledonous plants, including tomato, potato, and tobacco. Some crop species cannot be regenerated from leaf disks, and somaclonal variation may happen during shoot regeneration, which is one of this method's drawbacks. To address these issues, somaclonal variance is decreased and regeneration efficiency is increased by using shoot apices as explants. Agrobacterium inoculation has also been applied to other explants, including calli, shoot tips, hypocotyls, cotyledons, seedlings, and somatic embryos. While the process was primarily restricted to dicotyledonous plants, advances in tissue culture techniques have made it possible to successfully change monocotyledons as well.

Challenges and Potential Solutions

Agrobacterium-mediated transformation is widely used, although its effectiveness is limited by a number of issues. The target plant species plays a critical role in the transformation process's effectiveness since certain species naturally resist Agrobacterium infection. When compared to dicotyledonous plants, monocotyledonous plants in particular typically exhibit more resistance. Because different Agrobacterium strains have varying degrees of effectiveness in transforming plants, selecting a strain has a substantial impact on transformation efficiency as well. The final result of the transformation can also be influenced by other variables, including the concentration of Agrobacterium utilized, the length of co-cultivation, and the selection of selection markers and reporter genes.

Moreover, transgenic expression may be impacted by the surrounding native DNA sequences, and the integration of T-DNA into the plant genome is frequently unpredictable. There is a limit to the size of T-DNA that can be transferred, which also limits the quantity of genes or DNA fragments that can be added. The procedure of choosing particular features in genetically modified plants is made more difficult by the unpredictable character of T-DNA fusion.

Conclusion

Plant biotechnology still relies heavily on agrobacterium-mediated transformation, which has many benefits for genetic engineering. For this strategy to remain successful, it is necessary to address the problems that it faces. Technological developments in biotechnology, especially the creation of instruments such as CRISPR/Cas9, present viable ways to improve the accuracy and yield of Agrobacterium-mediated transformation. By overcoming these obstacles, scientists will be able to fully utilize this approach and open the door for the next generation of genetically modified crops, which will be more durable, productive, and able to fulfill future food demands worldwide.

Reference:

Rahman, S.U., Khan, M.O., Ullah, R. et al. Agrobacterium-Mediated Transformation for the Development of Transgenic Crops; Present and Future Prospects. Mol Biotechnol 66, 1836–1852 (2024). https://doi.org/10.1007/s12033-023-00826-8

Image Reference:

http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Agrotrans.GIF