Recent advances in Pelargonium in vitro regeneration systems

Recent advances in the development of protocols for in vitro culture and genetic manipulation have provided new avenues for the development of novel varieties of Pelargonium and for use as model systems for investigating the factors controlling plant morphogenesis. Optimized techniques of meristem culture have supplemented the culture indexing methods in commercial greenhouse production resulting in availability of large-scale pathogen indexed planting material. Currently, technologies are available for the mass in vitro propagation of F1 hybrid Pelargonium through both organogenesis and somatic embryogenesis. The somatic embryogenesis model system has allowed researchers to identify critical factors controlling plant morphogenesis in vitro such as regulation of regeneration by growth regulators, choice of explant and characterization of induction and expression phases of morphogenesis in Pelargonium. Also, optimization of technologies for genetic transformation of Pelargonium opened up the possibilities for developing genotypes with novel characters, including resistance to some of the major diseases. Finally, the development of regeneration systems for Pelargonium spp. has facilitated conventional crop improvement programs, thereby providing a valuable resource to the horticultural industry.     

Transfer of Dicamba Tolerance from Sinapis arvensis to Brassica napus via Embryo Rescue and Recurrent Backcross Breeding

Auxinic herbicides (e.g. dicamba) are extensively used in agriculture to selectively control broadleaf weeds. Although cultivated species of Brassicaceae (e.g. Canola) are susceptible to auxinic herbicides, some biotypes of Sinapis arvensis (wild mustard) were found dicamba resistant in Canada. In this research, dicamba tolerance from wild mustard was introgressed into canola through embryo rescue followed by conventional breeding. Intergeneric hybrids between S. arvensis (2n = 18) and B. napus (2n = 38) were produced through embryo rescue. Embryo formation and hybrid plant regeneration was achieved. Transfer of dicamba tolerance from S. arvensis into the hybrid plants was determined by molecular analysis and at the whole plant level. Dicamba tolerance was introgressed into B. napus by backcrossing for seven generations. Homozygous dicamba-tolerant B. napus lines were identified. The ploidy of the hybrid progeny was assessed by flow cytometry. Finally, introgression of the piece of DNA possibly containing the dicamba tolerance gene into B. napus was confirmed using florescence in situ hybridization (FISH). This research demonstrates for the first time stable introgression of dicamba tolerance from S. arvensis into B. napus via in vitro embryo rescue followed by repeated backcross breeding. Creation of dicamba-tolerant B. napus varieties by this approach may have potential to provide options to growers to choose a desirable herbicide-tolerant technology. Furthermore, adoption of such technology facilitates effective weed control, less tillage, and possibly minimize evolution of herbicide resistant weeds.     

Focus on Weed Control: Tandem Amplification of a Chromosomal Segment Harboring 5-Enolpyruvylshikimate-3-Phosphate Synthase Locus Confers Glyphosate Resistance in Kochia scoparia

Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations.Glyphosate [N-(phosphonomethyl) Gly] is the most widely used agricultural pesticide globally (Duke and Powles, 2008). Originally, being a nonselective herbicide, its use was limited to vegetation management in noncrop areas; however, introduction of glyphosate-resistant (GR) crops in the late 1990s, coupled with their exceptional adoption, led to accelerated use totaling approximately 128 million ha worldwide in 2012 (James, 2012). GR crop technology has made a significant contribution to global agriculture and the environment, as it not only increased farm income by $32.2 billion (Brookes and Barfoot, 2013), but also moderated the negative environmental impacts of mechanical weed management practices (Gardner and Nelson, 2008; Bonny, 2011). Glyphosate offers a simple, effective, and economic weed management option in GR crops. In addition, it provides immense value in no-till crop production systems by enabling soil and moisture conservation. However, due to intensive glyphosate selection pressure, several weed populations globally have evolved resistance through a variety of mechanisms. Globally, herbicide resistance, in particular the recent proliferation of glyphosate resistance in weed species, is a major crop protection threat; nearly two dozen GR weed species have been reported in the last 15 years (Heap, 2014).Glyphosate, an aminophosphonic analog of the natural amino acid Gly, nonselectively inhibits 5-Enolpyruvylshikimate-3-Phosphate synthase (EPSPS) in plants, preventing the biosynthesis of the aromatic amino acids Phe, Tyr, and Trp (Steinrücken and Amrhein, 1980), resulting in the death of glyphosate-sensitive individuals. In plants, EPSPS is one of the key enzymes in the shikimate pathway (Herrmann and Weaver, 1999), and glyphosate inhibits EPSPS by binding to EPSPS-shikimate-3-P binary complex forming an EPSPS-shikimate-3-P-glyphosate complex (Alibhai and Stallings, 2001). Bradshaw et al. (1997) hypothesized against the likelihood of weeds evolving resistance to glyphosate, primarily because of its complex biochemical interactions in the shikimate pathway and also due to the absence of known glyphosate metabolism in plants. Nonetheless, several cases of glyphosate resistance, as a result of difference in glyphosate translocation (Preston and Wakelin, 2008) or mutations in the EPSPS, were confirmed (Baerson et al., 2002). More importantly, duplication/amplification of the EPSPS appears to be the basis for glyphosate resistance in several weeds (Sammons and Gaines, 2014). Here, we use duplication to refer to the formation of first repetition of a chromosomal segment and amplification to refer to increase in number of the repetitions (more than two repetitions of a chromosomal segment) under positive selection. The first case of EPSPS amplification as a basis for glyphosate resistance was reported in an Amaranthus palmeri population from GA (Gaines et al., 2010). In this A. palmeri population, there is a massive increase (>100-fold relative to glyphosate-susceptible [GS] plants) in EPSPS copies, and these copies are dispersed throughout the genome (Gaines et al., 2010).Field-evolved GR Kochia scoparia populations were first reported in western Kansas in 2007 (Heap, 2014). We previously determined that evolution of GR populations of K. scoparia in the U.S. Great Plains is also due to amplification of the EPSPS (A. Wiersma and P. Westra, unpublished data). Unlike in GR A. palmeri, we found relative EPSPS:acetolactate synthase (ALS) copies ranging from three to nine in GR K. scoparia populations. While it quickly became widespread in the region, its presence was reported in another five Great Plains states by 2013 (Heap, 2014). GR K. scoparia populations we tested were 3- to 11-times resistant (population level) to glyphosate compared with a GS population (Godar, 2014), and EPSPS expression positively correlated with genomic EPSPS copy number (A. Wiersma and P. Westra, unpublished data). Here, we reveal the genomic organization of the amplified EPSPS copies in two GR K. scoparia populations, an alternative mechanism of gene amplification to that reported in GR A. palmeri.