Long-term T-DNA insert stability and transgene expression consistency in field propagated sugarcane.

KEY MESSAGE: This study addresses T-DNA insert stability and transgene expression consistency in multiple cycles of field propagated sugarcane. T-DNA inserts are stable; no transgene rearrangements were observed. AmCYAN1 and PMI protein accumulation levels were maintained. There was no evidence that production of either protein declined across generations and no transgene silencing was observed in three commercial sugarcane varieties through commercially relevant ratooning, propagation-by-setts, and micro-propagation generation processes over 4 years of field testing. Long term transgene expression consistency and T-DNA insert stability can be achieved in sugarcane, suggesting that it is highly probable that transgenic sugarcane can be successfully commercialized. This study addresses T-DNA insert stability and transgene expression consistency in multiple cycles of field propagated sugarcane. These data are critical supporting information needed for successful commercialization of GM sugarcane. Here seventeen transgenic events, containing the AmCYAN1 gene driven by a CMP promoter and the E. coli PMI gene driven by either a CMP or Ubi promoter, were used to monitor T-DNA insert stability and consistency of transgene encoded protein accumulation through commercially relevant ratooning, propagation-by-setts, and micro-propagation generation processes. The experiments were conducted in three commercial sugarcane varieties over 4 years of field testing. DNA gel blot analysis showed that the T-DNA inserts are stable; no transgene rearrangements were observed. Quantitative ELISA showed no evidence of decreasing AmCYAN1 and PMI protein levels across generations and no transgene silencing was observed. These results indicate that long term transgene expression consistency and T-DNA insert stability can be achieved in sugarcane, suggesting that it is highly probable that transgenic sugarcane can be successfully commercialized.

The structure, function, and biosynthesis of plant cell wall pectic polysaccharides.

Plant cell walls consist of carbohydrate, protein, and aromatic compounds and are essential to the proper growth and development of plants. The carbohydrate components make up approximately 90% of the primary wall, and are critical to wall function. There is a diversity of polysaccharides that make up the wall and that are classified as one of three types: cellulose, hemicellulose, or pectin. The pectins, which are most abundant in the plant primary cell walls and the middle lamellae, are a class of molecules defined by the presence of galacturonic acid. The pectic polysaccharides include the galacturonans (homogalacturonan, substituted galacturonans, and RG-II) and rhamnogalacturonan-I. Galacturonans have a backbone that consists of alpha-1,4-linked galacturonic acid. The identification of glycosyltransferases involved in pectin synthesis is essential to the study of cell wall function in plant growth and development and for maximizing the value and use of plant polysaccharides in industry and human health. A detailed synopsis of the existing literature on pectin structure, function, and biosynthesis is presented.

The Arabidopsis irregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan, which are essential for secondary cell wall integrity.

The secondary cell wall in higher plants consists mainly of cellulose, lignin, and xylan and is the major component of biomass in many species. The Arabidopsis thaliana irregular xylem8 (irx8) mutant is dwarfed and has a significant reduction in secondary cell wall thickness. IRX8 belongs to a subgroup of glycosyltransferase family 8 called the GAUT1-related gene family, whose members include GAUT1, a homogalacturonan galacturonosyltransferase, and GAUT12 (IRX8). Here, we use comparative cell wall analyses to show that the irx8 mutant contains significantly reduced levels of xylan and homogalacturonan. Immunohistochemical analyses confirmed that the level of xylan was significantly reduced in the mutant. Structural fingerprinting of the cell wall polymers further revealed that irx8 is deficient in glucuronoxylan. To explore the biological function of IRX8, we crossed irx8 with irx1 (affecting cellulose synthase 8). The homozygous irx1 irx8 exhibited severely dwarfed phenotypes, suggesting that IRX8 is essential for cell wall integrity during cellulose deficiency. Taken together, the data presented show that IRX8 affects the level of glucuronoxylan and homogalacturonan in higher plants and that IRX8 provides an important link between the xylan polymer and the secondary cell wall matrix and directly affects secondary cell wall integrity.