Molecular and Functional Characterization of GR2-R1 Event Based Backcross Derived Lines of Golden Rice in the Genetic Background of a Mega Rice Variety Swarna

Abstract

Homozygous Golden Rice lines developed in the background of Swarna through marker assisted backcross breeding (MABB) using transgenic GR2-R1 event as a donor for the provitamin A trait have high levels of provitamin A (up to 20 ppm) but are dwarf with pale green leaves and drastically reduced panicle size, grain number and yield as compared to the recurrent parent, Swarna. In this study, we carried out detailed morphological, biochemical and molecular characterization of these lines in a quest to identify the probable reasons for their abnormal phenotype. Nucleotide blast analysis with the primer sequences used to amplify the transgene revealed that the integration of transgene disrupted the native OsAux1 gene, which codes for an auxin transmembrane transporter protein. Real time expression analysis of the transgenes (ZmPsy and CrtI) driven by endosperm-specific promoter revealed the leaky expression of the transgene in the vegetative tissues. We propose that the disruption of OsAux1 disturbed the fine balance of plant growth regulators viz., auxins, gibberellic acid and abscisic acid, leading to the abnormalities in the growth and development of the lines homozygous for the transgene. The study demonstrates the conserved roles of OsAux1 gene in rice and Arabidopsis.

Risk assessment of genetically engineered plants that can persist and propagate in the environment

“…In other cases, lower fitness is caused by unintended effects such as interruption of endogenous genes. For example, after crossing lines of "Golden Rice" with the Indian variety, Swarna, Bollinedi et al [28] observed a growth disturbance since the gene constructs encoding traits for increased production of precursors for vitamin A interfered with the plant's own gene for producing growth hormones. The integration of the transgene disrupted the native OsAux1 gene, which encodes an auxin transmembrane transporter protein, therefore most likely interfering with the fine-tuning of plant growth regulators auxin, gibberellic acid and abscisic acid.…”

“…The integration of the transgene disrupted the native OsAux1 gene, which encodes an auxin transmembrane transporter protein, therefore most likely interfering with the fine-tuning of plant growth regulators auxin, gibberellic acid and abscisic acid. The gene constructs were not, as intended, active solely in the kernels but also in the leaves [28]. This caused a substantial reduction in the chlorophyll content in the leaves, leading to pale green leaves in homozygous plants.…”

Limits of Knowledge and Tipping Points in the Risk Assessment of Gene Drive Organisms

“…It was concluded that existing experience was in line with the above hypothesis. Next generation effects Next generations of GE organisms can show effects that were not observed or intended in the original event (Kawata et al 2009;Cao et al 2009;Yang et al 2017) Effects emerging from genetic background Unintended effects can emerge from interaction of the newly inserted genes with the genetic backgrounds (Bollinedi et al 2017;Lu and Yang 2009;Vacher et al 2004;Adamczyk and Meredith 2004;Adamczyk et al 2009) Interaction with the environment on the level of the genome (genome × environment interactions)…”

Golden Rice: To Combat Vitamin A Deficiency for Public Health

“…The agronomy of Golden Rice-how it grows, its resistance to pests and diseases, its water requirements and days to maturity and plant and grain morphologies-and yield are the same as the variety into which the nutritional trait has been introduced. An avoidable human error was made in an earlier selection of 'a lead transformation event: GR2R' , which led to plants in open fields falling over when subject to wind and rain, and a small yield loss of about 2% was the result [9,38]. GR2R was dropped from development in late 2013.…”

“…Eighty percent-about 380 million tonnes-of global rice production is produced on small farms for family consumption, usually unprocessed except for polishing [38]. It is probably not stored for long, as rice is produced, usually, in two or three growth cycles annually, and storage facilities are limited.…”