P HYSICAL M EASUREMENTS

Physical measurements consist in average fresh weight determination and tissue proportion analysis, dry matter content and dry weight calculation.

2.4.1 F

W

D

At each sampling date 10 fruit [unless total plant fruit was lower than (10Xsampling date number)] were singularly weighed on a two decimal position electronic scale and the weight recorded.

2.4.2 T

P

At each sampling date fruit a picture of the whole fruit, a transversal and a longitudinal section were taken using a digital camera (Nikon, Coolpix 990). For early sampling dates (up to 2 WAFB) picture were taken using the camera on a stereo-microscope (Nikon, SMZ1500). As ruler was placed as scale in each picture.

2.4.2.1 IMAGE ANALYSIS

Images were analyzed using ImageJ (Rosband, 1997-2006). The plugins option ‘Analyze:

Area Calculation’ was used.

2.4.3 D

M

Each equatorial slice was oven-dried to a constant weight at 60-65^oC, and the dry weight of the slice recorded and expressed as a percentage of the fresh weight (DM (%)). The dehydration was carried out by an Ezidri^TM Home Food Dehydrator model Ultra FD 1000 from Hidraflow Industries Limited (NZ). The weight was determined using a 3 decimals scale (Mattler Toledo, PB303-S/FACT). Slices were placed on 60 mm diameter Petri dishes (Sarstedt, Cat. #821194).

2.4.4 D

W

C

Dry weight was calculated using the formula reported in Eq. 2-1 dry weight. (DW) and fresh weight (FW) were expressed in g, whereas dry matter (DM) was reported as percentage.

DW =

FW x DM

100

Eq. 2-1

2.5 A

2.5.1 S

P

L

M

Specimen preparation was carried out as for Hallett and Sutherland (2005) and it consists of 6 phases: fixation, dehydration, resin embedding and capsule inclusion-polymerization, block trimming and sectioning, slide staining and mounting.

2.5.1.1 FIXATION

Before start to process sample, excess fixative was poured into labelled vials (specimen name, date and fixative type). Fine forceps/tweezers and scalpel with fresh blade were also prepared.

As soon as fruit were collected a central 1-5 mm transversal slice was cut from each berry. A sector comprehensive of all three tissues was then cut off as shown in Fig. 2-2 and quickly immersed in the Glutaraldehyde-Formaldehyde fixative (2.5%-2%).

Fig. 2-2: Diagrammatic cross-section of a segment of kiwifruit. C = core, IP = inner pericarp, L = locule, LW = locule wall, OP = outer pericarp, S = skin layers (epicarp), V = location of major vascular trace (Hallett et al., 1992).

Fixative penetration (particularly for plant material) was aided by light evacuation (to pull out trapped air). Loosely capped vials were put in the vacuum desiccator and a light vacuum was pulled using the water aspirator. When bubbles were generated the vacuum was close off, but it was kept running (about 1 hour). When the vacuum was released it was checked that samples were not floating on the fixative surface. Cap was then securely fastened.

Samples were than stored at 4ºC for 1-2 weeks.

2.5.1.2 DEHYDRATION

After fixation the samples were washed for 1 hour in 50 mM phosphate buffer (3 changes over 1 hour period using the rotator to agitate), rinsed quickly in distilled water, then replaced with 10% ethanol.

Dehydration was held with progressively more concentrated ethanol solutions to 100% 10-20 minutes per step using the rotator to agitate. Last step was a double change of 100% dried ethanol.

2.5.1.3 RESIN EMBEDDING

The last 100% ethanol was replaced with LR White Resin (London Resin, Reading, UK).

Vials were agitated on the rotator overnight. The second day, two changes were carried out.

Then sample resin was changed on each of the next two days.

2.5.1.4 C^APSULE INCLUSION AND POLYMERIZATION

A small label with specimen number and LR White Resin were placed into gelatine capsules, the specimen was quickly included and then the cap sealed. The resin was hardened by heat polymerization in embedding oven in fume hood at 60ºC for at least 8 hours. Then the blocks were let cool down and stored in a small labelled bag.

2.5.1.5 S^LIDE P^REPARATION:B^LOCK TRIMMING AND S^ECTIONING

Block surface was trimmed first with a sharp blade to a trapezium shape surface. A more fine trimming was performed with a glass knife at the microtome (Leica, UltraCut UCT). 10 sections were then cut 1 µm thick, collected with a loop and placed on a drop of water on a glass slide. Slide was dried on a hot plate at 50ºC.

2.5.1.6 SLIDE PREPARATION:STAINING AND MOUNTING

Sections of resin-embedded material were stained in a 0.05% solution of toluidine blue in benzoate buffer (pH 4.4) for 5 minutes, 3 time water rinsed, air dried, and mounted in Shurmount (Triangle Biomedical Sciences, Durham, NC).

2.5.2 L

M

O

Sections were viewed using an Olympus Vanox AHT3 microscope (Olympus Optical, Tokyo), coupled to a CoolSnap (Roper Scientific Ltd, Tucson, Arizona) digital camera, and the image was projected on a computer screen using RSImage software (Roper Scientific Ltd, Tucson, Arizona) and the magnification recorded. Observations were held from 40X to 400X magnifications. A reference scale picture was taken at each used magnification, for further measurements.

2.5.3 S

P

S

-M

Samples were collected, fixed and dehydrated as for §§ 2.5.1.1 and 2.5.1.2. After dehydration, samples were stained and pictures were taken as following reported.

2.5.3.1 STAINING TECHNIQUE FOR CELL COUNTING

The cell walls at the surface of each slice were stained as reported in (Goffinet et al., 1995) by immersing sectors for three minutes in 5% aqueous tannic acid, rinsing briefly in

tap-water, and immersing in 1% ferric ammonium sulphate (iron alum) for one minute, then rinsed again in water.

2.5.4 S

-M

O

After a second water rinse the sectors were pinned to the bottom of water-filled Petri dishes and examined at a 6X magnification with a stereo-microscope (Nikon, SMZ1500). Images were obtained using a Camera Control Unit (Nikon, SU-1) and analyzed by using ImageJ software (Rosband, 1997-2006). Before start each set of image captures, a picture of a scale was taken as zoom camera reference (Maiji Techno, 1 mm in 100 divisions).

2.6 B

T