Selection Without Using Antibiotic Resistance Genes in Chloroplast Transformation

Introduction

Genes conferring antibiotic resistance have been widely used as markers for selection of transformed cells during plant chloroplast transformation. But the presence of these antibiotics in genetically modified plants, released in the environment or used as food or feed, has raised concerns about possible risks to human health and the environment. Betaine aldehyde dehydrogenase (BADH) localizes to different subcellular regions, including the chloroplast, peroxisome, or cytosol. The study found that the BADH gene family encodes a diverse set of multifunctional proteins that are catalyzed by different mechanisms. Cloning and enzymatic activity studies of BADH in plants were initially performed in spinach. With the advancement of molecular biotechnology, studies have found that the BADH gene is multifunctional and plays an important role in aroma production, abiotic stress, and antibiotic-free selection of transgenic plants. The scientists' BADH isolated from spinach was successfully used to select for chloroplast transformation of tobacco to prevent the risk of transferring antibiotic resistance genes into gut microbes or the environment.

Fig. 1. The function of BADH in the tolerance to abiotic stresses via the MAPK signalling pathway, and also metabolic synthesis of glycine betaine in plants. (Ozyigit II, et al., 2020)

Our Services

BADH is encoded by nuclear genes and transported to the action site in the chloroplast, where it converts the toxic betaine aldehyde into the nontoxic glycine betaine. Based on this feature, Lifeasible is committed to developing GMOs without the use of antibiotic resistance marker genes. We have successfully achieved rapid regeneration of chloroplast transgenic plants using BADH as a useful surrogate for antibiotic resistance genes as a selectable marker for transgenic plants. We can also analyze the role of BADH as a positive regulator in controlling plant responses to environmental stress and stress-related Metabolism, contributing to the rapid improvement of crop varieties. In addition, we offer a selection of other alternative antibiotic resistance gene markers.

· Plant-Derived Antibiotic Resistance Marker Genes
Some plant genes may also be involved in antibiotic resistance. We successfully overexpressed the A. thaliana DEF2 (peptide deformylase) and GPT (UDP-N-acetylglucosamine:dolichol phosphate N-acetylglucosamine-1-P transferase) genes in tobacco and Arabidopsis thaliana to confer actinomycin and Tunicamycin resistance.

· Marker Genes Conferring Herbicide Resistance
We offer many herbicide resistance genes from bacterial, fungal, plant and even mammalian sources for transformation of transgenic plants. This service targets transgenic plants tested in the field.

Advantages of Using BADH As Antibiotic Resistance Genes

• This strategy is only applicable to the chloroplasts of a few plants that grow in arid and saline environments.
• Compared with the use of antibiotic resistance marker genes of microbial origin, some of the associated biosafety issues can be bypassed.
• The regeneration speed of transformed plants is improved, and the screening time is shortened accordingly.
• Transfer of antibiotic genes from plants can be avoided, helping to alleviate public concern about genetic modification.
• A complete assessment of the overall biosafety of the selectable marker for each GM crop.

Lifeasible has successfully employed multiple strategies for chloroplast gene transformation without the use of antibiotic resistance genes. With our team's extensive experience in antibiotic-free chloroplast transformation, we are able to provide global customers with antibiotic-free resistance gene screening using the spinach betaine aldehyde dehydrogenase (BADH) gene as a marker. If you are interested in our services, please do not hesitate to contact us for more information.

References

1. Golestan Hashemi F S, Ismail M R, Rafii M Y, et al. (2018) Critical multifunctional role of the betaine aldehyde dehydrogenase gene in plants[J]. Biotechnology & Biotechnological Equipment. 32(4): 815-829.
2. Daniell H, Muthukumar B, Lee SB. (2001) Marker free transgenic plants: engineering the chloroplast genome without the use of antibiotic selection. Curr Genet. 39(2):109-16.