Análisis de la información por categorías e interpretación de los datos

7 1

Several fruit were collected at 8 5 and 1 4 1 DAFB for microscopic study. Fruit from both collection dates were infiltrated with dye and cut transversally into three, approximately equal, disks (using a sharp knife): disc 'A' was from near the stalk-end of the fruit, 'B' was from the middle and 'C' from near the calyx-end. In each disc, randomly chosen dysfunctional (unstained) bundle was marked on the skin along assumed path and excised with a small amount of attached flesh so as to produce an elongated sample. Fruit picked at 85 DAFB were used to identify structural changes in the xylem. Each sample was soaked for several minutes in an excess of phloroglucin solution (2.5 g in 5 0ml of 70% v/v aqueous ethyl alcohol) and flooded afterwards with concentrated HCI to visualise lignified vascular elements. This procedure yields distinctively red-stained xylem strands embedded within a reasonably transparent parenchyma. To avoid rupture of xylem during the final tissue preparation, the bulk of the flesh was carefully dissected away with a microtome blade and fine forceps working under a dissecting microscope (Wild M3Z). The excised bundle was mounted in 80% w/w glycerol on a glass slide and examined with a compound light microscope (Microlux-l l) attached to a video-camera system.

Fruit picked at 1 4 1 DAFB were used to investigate the nature of xylem disruption by a more sophisticated light microscopic procedure. To prepare dysfunctional bundle for fixation, intercellular air was first removed by evacuation. To do this the sample was

placed in a 50 cm3 vial containing water and wetting agent (0. 1 % w/w Silwet L-77), and

exposed to reduced atmospheric pressure (c. 1 8 mmHg at 20°C) for 2 min. After the

vacuum had been released, water flowed in to replace the extracted air over a 2 min period. This treatment rendered the parenchyma reasonably transparent and suitable for dissection. Samples were trimmed with a sharp blade under the dissecting microscope (Wild M3Z) until the bundle was exposed with a very thin layer of surrounding flesh. It was then cut transversally every 4 - 5 mm. A total of five, four and three specimens were

obtained from disks A, B and C respectively.

Once ordered, (AI to As) the specimens were preserved separately in sealed glass vials containing half-strength Karnovsky's fixative (3% v/v glutaraldehyde and 2% w/v formaldehyde in 0. 1 M phosphate buffer at pH 7.2). Secondary fixation was in 1 % osmium tetroxide using the same buffer. Specimens were dehydrated in a graded acetone series, and infiltrated and embedded in Procure 8 12 epoxy resin. Cured resin blocks were trimmed and longitudinal sections ( l /-tm thick) were cut from the middle of the bundle, heat mounted on glass microscope slides and stained with 0. 1 % toludine blue. Tissues were prepared for microscopy and sections by Mr Douglas Hopcroft, EM Unit,

HortResearch, Palmerston North. Slides were examined and photographed using a Zeiss Axioplan compound light microscope with attached MC 1 00 camera system.

A view of xylem breakage in primary bundles was obtained from the same fruit used for photomicrograph examination ( 1 4 1 DAFB). Fruit were cut transversally (with a sharp knife) into three uniform slices and each was re-cut longitudinally (with a microtome blade) along the path of unstained bundles so as to expose regions where dye abruptly stopped. Specimens were photographed using a Wild M3Z dissecting microscope with attached 3 5 mm Leica-Wild MPS52 camera system.

4.2.6. Growth of the different fruit tissues

To evaluate the growth of the different fruit tissues, dimensional measurements relative to the axis were made in the fruit of both cultivars (Fig. 4.2). These were measured in the same fruit used for dye infusion. One extra fruit of each cultivar at each time was cut equatorially using a microtome blade. Measurements were made using digital callipers (precision 0.01 mm).

Chapter 4. Causes and effects of changes in xylem functionality in apple fruit ₇₃

Radial distances were taken from the fruit axis to the skin through the primary (sepal and petal) bundles. These gave a measure of the absolute tissue growth (ATG) of the core and cortex (assuming that the vascular ring delineates the boundary between two fleshy portions ofthe fruit) and of the fruit itself. The length of the cartilaginous walls defining locules was also recorded. The dimension of the fleshy region between the tip of the locule and the adjacent sepal bundle was obtained by difference. Relative tissue growth (RTG) was obtained by normalising the dimensional tissue measurements with the corresponding fruit radii to determine how the various structural components contributed to the growth of the whole.

S

P 1

L

Fig. 4.2. Transverse section of fruit showing radial dimensions measured from the fruit axis to the skin. Here S I refers to sepal bundle whereas P I refers to petal bundle. Hence, distance from the fruit axis to Ll gives a measure of locule, to S 1 and P I gives a measure of core (ovary) and to S l ' and P I ' gives a measure of fruit dimensions respectively. The difference between fruit and core dimensions quantifies the size of the cortex. Following this approach, five locule dimensions and ten core, cortex and fruit dimensions were obtained for each fruit. In addition, the distance between the locule tip (Ll ) and the sepal bundle was assessed.

Observations of vascular functionality made about midway through the season revealed that the sepal bundles of the cortical vascular system of ' Granny Smith' retained

functionality for longer than the petal bundles. This distinction was not apparent in 'Braeburn' . The spatial arrangements of the petal and sepal bundles were assessed in both cultivars. Each point was defined by the ratio of the distances of the petal and sepal bundles to the fruit axis (ten individual measurements) and so it was necessary to carry out analysis by assessing the mean ratio for a single fruit. For each cultivar PROC GLM (SAS, 2000) was used to evaluate the significance of the slope of the mean ratio of the radial distances to petal and sepal bundles in the fruit (PIS) vs DAFB.

It seemed reasonable to assume that the degree of sepal bundle drift outward was related to the magnitude of ovary growth in the same direction. This emphasised the importance of the proximity of the sepal bundle to the neighbouring locule (S-L) normalised with the corresponding fruit radii. The significance of cultivar differences in the time course for the mean ratio was compared using PROC GLM (SAS, 2000).