This section contains specific details of materials and methods for studies included in this chapter. General materials and methods also relevant to this chapter are described in Chapter 2.
3.2.1 Plant material and growth conditions
Details of plants and plant growth conditions used for the experiments presented in this chapter are outlined in Table 3.1. Plants were either grown in the UTAS phytotron with total photoperiod comprising a base photoperiod of 8 hours of natural daylight extended with fluorescent light for longer photoperiods, or in controlled environment growth cabinets at 20°C under fluorescent light for the full photoperiod. Plants for characterisation of ontogenetic variation in internode length and branching in LD and SD for Sections 3.3.1.2-3.3.2 were grown at the same time, to enable comparison between photoperiods. Plants for the SD expression series in
gigas and wild-type for qRT-PCR analysis presented in Section 3.3.1.3 were grown and harvested by V. Hecht (Hecht et al., 2011).
Due to sterility of veg1 and veg2-1 mutants under all known conditions (Murfet and Reid, 1993), samples from these mutants were obtained by growing segregating populations. As veg1 and veg2-1 are gene deletion mutants (Berbel et al., 2012; see Chapter 4), there was no practical means of identifying plants that were heterozygous at these loci, from those that were homozygous, prior to growing their seed. Thus to obtain mutants, seed was grown from wild-type siblings from previous generations, which yielded wild-type families and segregating families. Glasshouse space restrictions limited the number of plants that could be grown at any one time, and the proportion of mutant segregants was sometimes less than expected, so mutant numbers were often limiting for experiments. Seed for gigas mutants was easily obtained from mutants previously grown under SD conditions, wherein these mutants flower and set seed (Murfet, 1992). Due to incomplete introgression of veg1 and
veg2-1 on to a wild-type line NGB5839 background, wild-type siblings from several different wild-type families were used as the wild-type line for these mutants.
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Table 3.1. Details of plant material and growth conditions for experiments presented in Chapter 3. For expression studies, tissue type and time-points of tissue harvest (days after sowing) are indicated. Tissues harvested include: main shoot apex (A), apex of the longest branch (B), or secondary inflorescence stub (I2S) or apex (I2A). For developmental series the range of time-points is shown with
the number of time-points indicated in parentheses. Number of plants (n) is shown. For expression studies, n represents each time-point.
Purpose Growth
conditions
Chapter
section(s) Tissue Genotypes
Time- point (days) n Expression of molecular markers for meristem identity 8h SD (Phytotron) 3.3.1.3 3.3.3 A + B combined WT (NGB5839) 81 2 gigas-2 81 112 2 3 veg2-2 81 112 3 3 WT (veg2-1) 81 2 veg2-1 81 1 WT (veg1) 81 2 veg1 81 2 8h SD (Cabinet) 3.3.1.3 A WT (NGB5839) 7-56 (8) 4 gigas-2 7-105 (10) 4 18h LD (Phytotron) 3.3.1.1 3.3.3 A (+B where A limiting 74d) WT (NGB5839) 45 3 gigas-2 45 3 veg2-2 45 74 3 3 WT (veg1) 45 3 veg1 45 74 2 2 WT (veg2-1) 45 3 veg2-1 45 74 3 3 3.3.4 I2S WT (NGB5839) 59 2 I2A veg2-2 74 3 24h LD (Cabinet) 3.3.1.1 A WT (NGB5839) 14 35 4 4 Characterisation of ontogenetic variation in internode length and branching 8h SD (Phytotron) 3.3.1.3 3.3.2 WT (NGB5839) 6 gigas-2 6 veg2-2 6 WT (veg2-1) 6 veg2-1 3 WT (veg1) 6 veg1 6 24h LD (Phytotron) 3.3.1.2 3.3.2 WT (NGB5839) intact 6 WT (NGB5839) deflowered 6 gigas-2 6 veg2-2 6 WT (veg2-1) 6 veg2-1 5 WT (veg1) 6 veg1 6 Characterisation of veg2-2 floral morphology 24h LD (Phytotron) 3.3.5.1 veg2-2 7 8h SD (Phytotron) 8 Characterisation of pim veg2-2 phenotype 18h LD (Phytotron) 3.3.5.2 WT (NGB5839) 20 veg2-2 17 pim-2 14 pim-2 veg2-2 23
67 Page 67 3.2.2 Genotyping
veg1 and veg2-1 mutants were identified by genotyping plants in segregating populations with PCR-based markers. Putative veg1 mutants were identified by absence of PCR product from primer pair PsFULc-2F and PsFULc-2R (see Appendix 1), which amplify a 950-bp fragment from the VEG1 gene in homozygous wild-type and heterozygous plants (Berbel et al., 2012). Putative veg2-1 mutants were identified by absence of PCR product from primer pair PsFD-7F and PsFD-6R, which amplify a 1.2kb fragment from the FDa gene in homozygous wild-type and heterozygous plants. Identity of putative mutants was confirmed by growing plants until appearance of distinctive aerial branching phenotype. Putative mutants used for harvest of material at early time-points for expression analysis, were kept until confirmation of identity by plant phenotype before apical samples were included in expression experiments.
Prior to characterisation of pim-2 veg2-2 double mutant phenotype, double mutant plants and single mutants heterozygous for the second mutant allele were identified using CAPS markers designed for both pim-2 and veg2-2 mutant alleles. For the PIM locus, the 185bp PCR product from primer pair PsPIM-F1 and PsPIM- R1 was digested with XmnI (New England Biolabs, Inc., Ipswich, MA), according to the manufacturer’s instructions. This enzyme digests PCR product containing the
pim-2 mutation, yielding 19bp and 166bp digestion products, but does not cut wild- type PCR products. For the VEG2 locus, the 732bp PCR product from primer pair PsFD-4F and PsFD-5R was digested with BspHI (New England Biolabs, Inc., Ipswich, MA), according to the manufacturer’s instructions. This enzyme digests PCR product containing the veg2-2 mutation, yielding 246bp and 486bp digestion products, but does not cut wild-type PCR products. For this study, plants were grown only from single mutants heterozygous for the second mutant allele, and double mutants were identified by distinctive phenotype.
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3.2.3 qRT-PCR analysis
For expression experiments included in Sections 3.3.1, 3.3.3 and 3.3.4, expression levels of DET, VEG1 and PIM relative to ACTIN were measured by qRT- PCR (see Appendix 1 for primer details). Samples comprised dissected apical buds (2mm2; main shoot apex, branch apex, or indeterminate apex of the veg2-2 I2,) or I2 stubs (approximately 1cm of tissue from the last I2 node to stub tip) as indicated in figure legends. For growth conditions and time-points, see Table 3.1. Time-points for
DET expression experiments included in Sections 3.3.1.1 and 3.3.1.3 were chosen to coincide with the expected peaks in DET expression for wild-type and veg2-2 plants, which corresponded to approximately one week after the first macroscopic appearance of developing flower buds in the apex of each genotype, as determined from preliminary expression studies (Sussmilch, 2008; see also Chapter 5). The SD
gigas expression series shown in Section 3.3.1.3 was harvested and processed with expression of ACT in RT- and RT+ and expression of DET on a single replicate determined by V. Hecht, prior to this study. During the time-frame of this study,
DET expression on a second replicate of these existing samples was determined, and all other experiments were conducted without technical assistance.
3.2.4 Plant measurements
For characterisation of ontogenetic variation in internode length for Sections 3.3.1.2 and 3.3.1.3, internode length was measured for all nodes on the stem with expanded leaves present at time of harvest of dried wild-type plants (LD: 97 days after sowing; SD: 152 days after sowing). To determine days to first open flower (DTF), plants were checked for flowers every 1-3 days, and stage of flower development was used to estimate date of flower opening.
To characterise branching during development, the total length and number of nodes were measured for the main lateral present at each stem node, in LD and SD conditions. Six weekly time-points covered early branch outgrowth and establishment of the aerial branching phenotype in the mutants (36-71 days after sowing in LD; 64-99 days after sowing in SD). A final seventh time-point was taken at plant harvest after seed maturation and drying in the wild-type plants (97 days after sowing in LD; 152 days after sowing in SD). The increase in length and number of nodes was calculated by subtracting the measurement of the previous time-point
69 Page 69 from each new measurement, separately for each lateral. Decrease in lateral length
due to drying was considered to represent a 0mm increase in lateral length. Mean values for each node were calculated for each genotype. I2 structures were not included in lateral measurements. Data for the first time-point for branching measurements under LD conditions (36 days after sowing) is not included for veg2-2. In order to characterise timing of lateral outgrowth, two arbitrarily defined stages of this process were investigated in the same experiment described above. The length of each lateral on the main stem was measured each week from the first time- point (LD: 36 days after sowing; SD: 64 days after sowing) until the first ‘enlarged aerial bud’ (axillary bud ≥5mm at node 8 or above) and the first ‘aerial branch’ (lateral with one or more expanded leaves at node 8 or above) had developed. Some plants had not shown aerial lateral outgrowth by these measures before the time of plant harvest (LD: 97 days after sowing; SD: 152 days after sowing). If multiple nodes had developed enlarged aerial buds or aerial branches between time-points, the node with the longest lateral was counted as the node of first enlarged aerial bud or aerial branch, respectively. Mean plant age at time of aerial bud outgrowth or branch development was calculated using the midpoint of measurement time-point intervals. The first interval was defined as ranging from 21 days after sowing until the first branching measurement time-point (LD: 36 days after sowing; SD: 64 days after sowing) to account for the time required for plants to develop 8 expanded nodes.
Measurements for comparison of total branching included all vegetative laterals that were 5mm or longer in length. The portion of each indeterminate I2 structure in veg2-2 after the I2 structure had reverted to I1 appearance was also included in measurements of total branching. For this trait, laterals and the main stem were measured at time of plant harvest (LD: 97 days after sowing; SD: 152 days after sowing).
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