General Aspects

Antibiotic resistance as a selection system

Plant biotechnology is based on the delivery, integration and expression of defined genes into plant cells, which can be grown to generate transformed plants. Efficiency of stable gene transfer is not high even in the most successful transfer systems and only a fraction of the cells exposed integrate the DNA construct into their genomes. Moreover, a successful gene transfer does not guarantee expression, even by using signals for the regulation of transgene expression. Therefore, systems to select the transformed cells, tissues or organisms from the non−transformed ones are indispensable to regenerate the truly genetically transformed organisms.

Antibiotic resistance genes allow transformed cells expressing them to be selected for out of populations of non−transformed cells. As part of this system, a selective toxic agent that interferes with the cellular metabolism is applied to a population of putatively transformed cells. The population of cells that has been transformed with and expresses a resistance gene is able to neutralize the toxic effect of the selective agent, either by detoxification of the antibiotic through enzymatic modification or by evasion of the antibiotic through alteration of the target.

The antibiotic resistance genes can be the genes of interest in their own right or they can be operatively linked to other genes to be transformed into the organisms.

Main components of an antibiotic resistance system

The effectiveness of a particular antibiotic resistance system depends mainly on the following elements:

  • The selective agent. It should fully inhibit growth of untransformed cells. The lowest concentration of the toxic compound that suppresses growth of the non−transformed cells and does not cause detrimental effects to the transformed ones is used.
  • The resistance gene. Transcriptional and translational control signals fused to the resistance gene determine to a great extent the expression level of resistance. In addition, the gene sequence plays an important role as some are more compatible with animal or plant systems or subgroups of animals and plants, such as monocotyledonous or dicotyledonous plants.
  • The material to be selected. In the case of plants, sensitivity to the selective agent depends on many factors, including the explant type, the developmental stage, tissue culture conditions and the genotype.

Apart from these factors, for an antibiotic resistance system to be efficient and useful the selectable marker gene should be expressible in a wide variety of cells and tissues, the background metabolic activity or resistance should be minimal or negligible, and a clear phenotypic change should be visible.

The most popular antibiotic resistance marker genes

Among the most widely used antibiotic resistance genes as selectable markers are neomycin phosphotransferase II (nptII) and hygromycin phosphotransferase (hpt). There are also other marker genes like gentamycin acetyltransferase (accC3) resistance and bleomycin and phleomycin resistance, but these are not as commonly used.

The enzyme NPTII inactivates by phosphorylation a number of aminoglycoside antibiotics such as kanamycin, neomycin, geneticin (or G418) and paromomycin. Of these, G418 is routinely used for selection of transformed mammalian cells. The other three are used in a diverse range of plant species, however, kanamycin has proved to be ineffective to select legumes and gramineae.

Hygromycin phosphotransferase is a suitable marker system for both plant and animal systems. The HPT enzyme inactivates the antibiotic hygromycin B. Hygromycin is usually more toxic than kanamycin and kills sensitive cells more quickly. It is nowadays one of the preferred antibiotic resistance marker systems for transformation of monocotyledonous plants, particularly gramineae (cereals and forages).