Tree Improvement Program Project Report 2006 / 2007

Dehydration: Long-shoot bud samples were
preprocessed for the stepwise dehydration series by a
series of four washes in a solution of 0.05 M Sorenson’s
phosphate buffer (pH 7.2) at room temperature. After
either the second or third wash, the buds were descaled
using tweezers and fine scissors.
Plant material was dehydrated by stepwise exposure to
an increased concentration of ethanol in water (30%, 50%,
70%, 95%, 100%). Distilled de-ionized water (ddH2O)
and anhydrous ethanol were used. Each step of the series
was left for a period of 1 h, with the exception of the final
step, which was left for 1.5 h.
Pre-infiltration: Pre-infiltration was carried out
according to the Technovit 7100 instructions. Preinfiltration
solution was made using a 1:1 ratio of Technovit
base to 100% ethanol. Dehydrated buds were immersed
in approximately 5 times their volume and left at room
temperature overnight.
Infiltration: Infiltration solution was made up
according to the Technovit 7100 instructions. One gram
of Hardener I was mixed into 100 mL Technovit base.
Buds were immersed in at least five times their volume of
infiltration solution. The solution was changed twice over
the period of infiltration: once to replace the pre-infiltration
solution and again on the third day of infiltration. After
the last infiltration solution change, the vials were stored in
refrigeration (4°C) until required.
Embedding: Embedding solution was made up
according to the Technovit 7100 instructions. Using a
ratio of 1 mL Hardener II to 15 mL infiltration solution,
the embedding solution was placed in embedding wells (3
cm diameter) using a Pasteur pipette. Bud specimens were
placed in the wells and covered with plastic film to allow
the solution to polymerize. This step took place at room
temperature.
Sectioning and staining: Sections (5 µm) were
obtained using a Leica SM2400 sledge microtome
equipped with a tungsten carbide knife. Each section
was manually flattened in distilled water on clean glass
slides (Technologist Choice), then stained with one of
the following stains: i) Toluidine blue O with sodium
hypochlorite pre-treatment, ii) Safranin O, or iii) Ponceau
2R with Azure II post-treatment (Gutmann et.al. 1996).
Slides were dried at 50°C on a slide warmer, then mounted
in Permount (Fisher Scientific) with clean glass cover slips
(VWR Scientific).
Sectioning strategy and avoidance of bias: To prevent
any bias occurring in the sectioning strategy, information
T R E E I M P R O V E M E N T P R O G R A M
P R O J E C T R E P O R T 2 0 0 6 / 2 0 0 7
concerning the properties of the original clones was
withheld from the investigators until the end. In addition,
the personnel who were trained to do the sectioning used
an initial practice set of a dozen samples, the results of
which are not included. In this way, sample processing
(fixation, dehydration, infiltration, and embedding) was
optimized and staff gained experience on appropriate
material without loss of critical samples. The total number
of samples sectioned was 109 (see Table 24). This represents
the labour of two individuals processing and sectioning
from September to mid-December. More samples were
sectioned from the 18 July to 15 August, 2006 collections
because this period proved to be the most critical.
Results
Female strobili appeared as large axillary buds with an apex
resembling that of a half dome. They were more easily
distinguishable in their later stages of development (Figure
68 and addendum). Bract primordia could be observed
along the proximal margin of the hemispherical apex. Scales
covering the developing strobilus originated at the base of
the bud.
The timing of female strobilus differentiation
varied between the different clones (Table 25). Female
differentiation was easiest to verify in genotypes that were
also high productive cone producers (Table 26). The best
example is 1540, in which only a few samples needed to be
sectioned to find female buds, largely because female buds
were abundantly available. In other samples, particularly
low-producing clones (1524, 1539), it was more difficult
to establish female differentiation, in spite of substantial
sample sectioning effort (Table 24).
Over the course of the summer, long-shoot buds
continued to expand, with the various cones differentiating
along their axes. There were clonal differences in longshoot
bud elongation over the course of the growing season
(Figure 69). The ones with the greatest elongation (1524
and 1539) appeared to have the most delayed female
development. The obverse was found with clones that had
the least elongation (1531 and 1540). One clone — 1506
— had slow elongation initially but increased its rate of
elongation, eventually matching the consistently faster
elongated clones. 1506 had early female development,
which may be linked to the slower initial elongation.
Those clones with the longest long-shoot buds were
from clones that historically had the least number of cones
per tree (Table 26). Assuming the long-shoot bud length is
consistent from year to year and that the cone production
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