Flowering (inflorescence formation) of the grass Lolium temulentum is strictly regulated, occurring rapidly on exposure to a single long day (LD). During floral induction, L. temulentum differs significantly from dicot species such as Arabidopsis in the expression, at the shoot apex, of two APETALA1 (AP1)-like genes, LtMADS1 and LtMADS2, and of L. temulentum LEAFY (LtLFY). As shown by in situ hybridization, LtMADS1 and LtMADS2 are expressed in the vegetative shoot apical meristem, but expression increases strongly within 30 h of LD floral induction. Later in floral development, LtMADS1 and LtMADS2 are expressed within spikelet and floret meristems and in the glume and lemma primordia. It is interesting that LtLFY is detected quite late (about 12 d after LD induction) within the spikelet meristems, glumes, and lemma primordia. These patterns contrast with Arabidopsis, where LFY and AP1 are consecutively activated early during flower formation. LtMADS2, when expressed in transgenic Arabidopsis plants under the control of the AP1 promoter, could partially complement the organ number defect of the severe ap1-15 mutant allele, confirming a close relationship between LtMADS2 and AP1.

Here, we report a form of oligonucleotide-directed mutagenesis for precision genome editing in plants that uses single-stranded oligonucleotides (ssODNs) to precisely and efficiently generate genome edits at DNA strand lesions made by DNA double strand break reagents. Employing a transgene model in Arabidopsis (Arabidopsis thaliana), we obtained a high frequency of precise targeted genome edits when ssODNs were introduced into protoplasts that were pretreated with the glycopeptide antibiotic phleomycin, a nonspecific DNA double strand breaker. Simultaneous delivery of ssODN and a site-specific DNA double strand breaker, either transcription activator-like effector nucleases (TALENs) or clustered, regularly interspaced, short palindromic repeats (CRISPR/Cas9), resulted in a much greater targeted genome-editing frequency compared with treatment with DNA double strand-breaking reagents alone. Using this site-specific approach, we applied the combination of ssODN and CRISPR/Cas9 to develop an herbicide tolerance trait in flax (Linum usitatissimum) by precisely editing the 59-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) genes. EPSPS edits occurred at sufficient frequency that we could regenerate whole plants from edited protoplasts without employing selection. These plants were subsequently determined to be tolerant to the herbicide glyphosate in greenhouse spray tests. Progeny (C1) of these plants showed the expected Mendelian segregation of EPSPS edits. Our findings show the enormous potential of using a genome-editing platform for precise, reliable trait development in crop plants.

Early changes in the concentrations of indole-3-acetic acid (IAA) and abscisic acid (ABA) were investigated in the larger axillary bud of 2-week-old Phaseolus vulgaris L. cv Tender Green seedlings after removal of the dominant apical bud. Concentrations of these two hormones were measured at 4,6,8,12 and 24 hours following decapitation of the apical bud and its subtending shoot. Quantitations were accomplished using either gas chromatography-mass spectrometry-selected ion monitoring (GC-MS- MATERIALS AND METHODS Plant MaterialsIn each set of experiments Phaseolus vulgaris L. cv Tender Green were grown in 30 x 60 x 10-cm plastic trays, filled with a 1:1 mixture of sand and peat moss, under a 16-h photoperiod at 23°C and 60% RH in a growth chamber (Controlled Environment Systems model PGV 36LT Winnipeg, Man.). Lighting was provided by high-pressure mercury lamps 250 ,uE m-2s-' PPFD. Fourteen days after planting, uniform seedlings were selected on the basis of height, leaf size, and size of both apical and axillary buds. These plants were randomly divided into groups of 60 plants (3 trays of 20 plants). In one-half of the plants the apical bud was removed by decapitation and in the other half the plants were left intact. Trays containing decapitated or intact plants were randomized in the chamber. At 2,4,6,8,12, and 24 h after decapitation the larger axillary bud was excised from each of the 60 plants for each of control and decapitated groups, and immediately immersed in liquid N2. All frozen tissues were lyophilized. Duplicate samples (each from 60 plants) of tissue were taken at each harvest for IAA and ABA analysis by GC-MS-SIM.2 In a second complete experiment axillary buds from another group ofplants were harvested solely for analysis of ABA by ELISA. The harvest times for examining ABA by ELISA were every 0.25 h up to 2 h, then every 0.5 h to 12 h, and hourly thereafter. The number of buds harvested for each ELISA was six, for each of decapitated and control plants.

Long-day exposure of the grass Lolium temulentum may regulate flowering via changes in gibberellin (GA) levels. Therefore, we have examined both GA levels and expression of a MYB transcription factor that is specific to the GA signal transduction pathway in monocots. This MYB gene from L. temulentum shows over 90% nucleotide identity with the barley and rice GAMYB genes, and, like them, gibberellic acid (GA 3 ) up-regulates its expression in the seed. Furthermore, cDNAs of both the barley and L. temulentum GAMYB show the same simple patterns of hybridization with digests of L. temulentum genomic DNA. Compared with vegetative shoot apices of L. temulentum, the in situ mRNA expression of LtGAMYB does not change during the earliest steps of "floral" initiation at the apex. However, by 100 h (the double-ridge stage of flowering) its expression increased substantially and was highest in the terminal and lateral spikelet sites. Thereafter, expression declined overall but then increased within stamen primordia. Prior to increased LtGAMYB expression, long-day exposure sufficient to induce flowering led to increased (5-to 20-fold) levels of GA 1 and GA 4 in the leaf. Thus, increases first in GA level in the leaf followed by increased expression of LtGAMYB in the apex suggest important signaling and/or response roles in flowering.

SummaryDifferences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide‐directed mutagenesis (ODM), one of the many tools of Cibus’ Rapid Trait Development System (RTDS ™) technology, offers a rapid, precise and non‐transgenic breeding alternative for trait improvement in agriculture to address this urgent need. This review explores the application of ODM as a precision genome editing technology, with emphasis on using oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of bacterial, fungal, mammalian and plant systems. The process of employing ODM by way of RTDS technology has been improved in many ways by utilizing a fluorescence conversion system wherein a blue fluorescent protein (BFP) can be changed to a green fluorescent protein (GFP) by editing a single nucleotide of the BFP gene (CAC→TAC; H66 to Y66). For example, dependent on oligonucleotide length, applying oligonucleotide‐mediated technology to target the BFP transgene in Arabidopsis thaliana protoplasts resulted in up to 0.05% precisely edited GFP loci. Here, the development of traits in commercially relevant plant varieties to improve crop performance by genome editing technologies such as ODM, and by extension RTDS, is reviewed.

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The genome of Brassica napus L. is the result of several polyploidization events that occurred during the history of B. napus. Due to its relatively short domestication history, diversity is relatively limited. An increasing number of loci in this crop’s genome have been gene-edited using various technologies and reagent delivery methods for basic research as well as for trait development. New alleles have been developed as edits in single, 2, 4, or more homologous loci in this important oilseed crop. This comprehensive review will summarize new alleles that have been developed as they relate to weed control, flowering, self-incompatibility, plant hormone biology, disease resistance, grain composition, and pod shatter reduction. These new alleles have significantly augmented our understanding of both plant growth and development for basic research as well as for their potential commercial impacts.

Knowing where and when different genes express at the shoot apex during the transition to flowering will help in understanding this developmental switch. The CDKA family of serine/threonine kinase genes are appropriate candidates for such developmental switching as they are involved in the regulation of the G1/S and G2/M boundaries of the cell cycle (see review by Dudits et al. 2007) and so could regulate increases of cell division associated with flowering. Furthermore, in rice stems the gibberellin (GA) class of plant growth regulators rapidly upregulate CDKA expression and cell division. Thus, CDKA expression might be linked to the florigenic action of GA as a photoperiodically-generated, signal. For the grass Lolium temulentum L., we have isolated an LtCDKA1;1 gene, which is upregulated in shoot apices collected soon after the start of a single florally inductive long day (LD). In contrast to weak expression of LtCDKA1;1 in the vegetative shoot apex, in situ and PCR-based mRNA assays and immunological studies of its protein show very rapid increases in the apical dome at the time that florigenic signals arrive at the apex (<6 h after the end of the LD). By ~54 h LtCDKA1;1 mRNA is localised to the floral target cells, the spikelet primordia. Later both LtCDKA1;1 mRNA and protein are most evident in floret meristems. Only ~10% of cells within the apical dome are dividing at any time but the LD increase in LtCDKA1;1 may reflect an early transient increase in the mitotic index (Jacqmard et al. 1993) as well as a later increase when spikelet primordia form. Increased expression of an AP1-like gene (LtMADS2) follows that of LtCDKA1;1. Overall, LtCDKA1;1 is a useful marker of both early florigenic signalling and of later morphological/developmental aspects of the floral transition.