Genetic and Global Epigenetic Modification, Which Determines the Phenotype of Transgenic Rice?

Abstract

Transgenic technologies have been applied to a wide range of biological research. However, information on the potential epigenetic effects of transgenic technology is still lacking. Here, we show that the transgenic process can simultaneously induce both genetic and epigenetic changes in rice. We analyzed genetic, epigenetic, and phenotypic changes in plants subjected to tissue culture regeneration, using transgenic lines expressing the same coding sequence from two different promoters in transgenic lines of two rice cultivars: Wuyunjing7 (WYJ7) and Nipponbare (NP). We determined the expression of OsNAR2.1 in two overexpression lines generated from the two cultivars, and in the RNA interference (RNAi) OsNAR2.1 line in NP. DNA methylation analyses were performed on wild-type cultivars (WYJ7 and NP), regenerated lines (CK, T0 plants), segregation-derived wild-type from pOsNAR2.1-OsNAR2.1 (SDWT), pOsNAR2.1-OsNAR2.1, pUbi-OsNAR2.1, and RNAi lines. Interestingly, we observed global methylation decreased in the T0 regenerated line of WYJ7 (CK-WJY7) and pOsNAR2.1-OsNAR2.1 lines but increased in pUbi-OsNAR2.1 and RNAi lines of NP. Furthermore, the methylation pattern in SDWT returned to the WYJ7 level after four generations. Phenotypic changes were detected in all the generated lines except for SDWT. Global methylation was found to decrease by 13% in pOsNAR2.1-OsNAR2.1 with an increase in plant height of 4.69% compared with WYJ7, and increased by 18% in pUbi-OsNAR2.1 with an increase of 17.36% in plant height compared with NP. This suggests an absence of a necessary link between global methylation and the phenotype of transgenic plants with OsNAR2.1 gene over-expression. However, epigenetic changes can influence phenotype during tissue culture, as seen in the massive methylation in CK-WYJ7, T0 regenerated lines, resulting in decreased plant height compared with the wild-type, in the absence of a transformed gene. We conclude that in the transgenic lines the phenotype is mainly determined by the nature and function of the transgene after four generations of transformation, while the global epigenetic modification is dependent on the genetic background. Our research suggests an innovative insight in explaining the reason behind the occurrence of transgenic plants with random and undesirable phenotypes.

Improving Nitrogen Use Efficiency Through Overexpression of Alanine Aminotransferase in Rice, Wheat, and Barley

“…Interestingly, it has been shown that Agrobacterium -mediated rice transgenic plants carried more unintended genomic mutations compared to particle bombardment or electroporation methods ( Labra et al, 2001 ; Wilson et al, 2006 ). More recently, Fan et al (2020b) reported the tissue culture process possibly causes epigenetic alterations in the rice genome. However, the stability of the epigenetic alterations may differ depending on the genetic background and may potentially be suppressed after some generations ( Fan et al, 2020b ).…”

“…More recently, Fan et al (2020b) reported the tissue culture process possibly causes epigenetic alterations in the rice genome. However, the stability of the epigenetic alterations may differ depending on the genetic background and may potentially be suppressed after some generations ( Fan et al, 2020b ). Those genomic alterations are heritable and independently segregate from GOI, which means it is possible that null segregants and GOI lines could carry different genomic profiles apart from the GOI.…”

Plant DNA methylation is sensitive to parent seed N content and influences the growth of rice

“…To confirm that the LPSN could influence the growth of OsNAR2.1 overexpression line, we also grown overexpression OsNAR2.1 line (Ov199) and OsNAR2.1 RNAi knock-down line (RNAi) of Nippobare background with wild type (NP) [ 25 , 26 ] with difference seed N content in four different N content field. The results showed that the plant height, grain yield, total N content of seeds and total N content of plant significantly decrease 22.3, 33, 50 and 38%, respectively, in Ov199-LN compared with Ov199-HN in HN field (Fig.…”

“…Nitrogen-deficiency stress can induce heritable alteration in DNA methylation [ 19 ]. Fan et al (2020) reported that overexpression of OsNAR2.1 does change rice DNA methylation status [ 25 ]. However, the research progress regarding epigenetic influence by the nutrient in both external and endogenous seed is very sparse.…”

Recent Advances in Molecular Research in Rice: Agronomically Important Traits

“…Gao et al identified that the OsEWL4 gene was a regulator of tiller number and plant biomass (yield of above-ground parts in rice plants) by using targeted mutations of eight rice FLW genes using the CRISPR/Cas9 system [ 11 ]. Fan et al produced overexpression and knockdown transformants of the OsNAR2.1 gene and revealed that changing expression levels of the OsNAR2.1 gene altered global methylation at the whole genome level and the phenotypes of plant height and grain yield at the plant level [ 12 ]. These novel technologies are powerful tools to understand the molecular basis of increasing rice yields.…”