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.