Definición de optimización

4.2.1

Fructification

4.2.1.1 Presence / absence of spadix

Stems (spathe plus spadix and peduncle) of Z. aethiopica were obtained from

plants grown in the display gardens of Massey University, Palmerston North. Stems were cut from plants either at:

 the unfurling bud-stage, when white colouration was appearing on the edge of the previously green-coloured buds, with the bud starting to unfurl, and with the male and female flowers still tightly wrapped by the spathe; or

 horticultural harvest-maturity, i.e., one to two days before pollen shed; spathe fully open and de-greened.

Stems of ‘Best Gold’ at horticultural harvest-maturity (i.e. 1-2 days before pollen shed, spadix turned corn-yellow, spathe fully coloured (Funnell and Downs, 1987)), were attained from plants grown in a heated glasshouse (growing conditions as described previously, refer Chapter 2 Section 2.2.1).

The spadix was either left intact or removed immediately after the stem was cut from the plant. For Z. aethiopica, due to the difficulty of removing the spadix

when the bud is tightly wrapped, the spadix was removed at the unfurling bud-stage.

The four individual stem replicates, for each treatment, were subsequently monitored for changes in colour of the spathe, while held in a vase-life room with reverse osmosis (RO) water supplied in glass jars. The vase-life room was controlled to environmental conditions recommended by Reid and Kofranek (1980): room temperature 20 ± 1 ºC, relative humidity 60 to 70%, 12-h (0600 HR – 1800 HR) photoperiod and light intensity of 25 µmol·m-2·s-1 at bench height provided by cool- white fluorescent tubes.

Measurement of colour changes of the abaxial surface of the spathe at basal, central and upper positions, were conducted as described in Chapter 2, which comprised recording the colour coordinates of lightness (L*), chroma (C*), and hue angle (Hº), using a tristimulus colorimeter CM-2600d/2500d (Konica Minolta, Japan). The measurement of colour was repeated every 2 to 4 days for a period of two weeks. A change in Hº can be used to describe the changes in chlorophyll content in the

abaxial surface of ‘Best Gold’, particularly during the initial stage of spathe re- greening (Chapter 2). While a similar correlation has not previously been established for Z. aethiopica, preliminary tests validated that Hº could be used to describe re-

greening of the spathe. A value of Hº greater than 90º has previously been found to be indicative of a level of chlorophyll visible to the human eye in the peel of Citrus

unshiu (Mak.) Marc. (Iglesias et al., 2001), and this criterion was also applied for the

abaxial surface of the spathe in ‘Best Gold’. Hence, in this experiment, for both ‘Best Gold’ and Z. aethiopica, the primary data of interest were Hº.

4.2.1.2 Presence / absence of female flowers

In some instances the spadix of ‘Best Gold’ is naturally devoid of the true female flowers, with only male flowers being present. This divergence in morphology was not observed in Z. aethiopica grown in the gardens of Massey University and,

therefore, in the current experiment only ‘Best Gold’ was studied for the influence of the presence or absence of female flowers on re-greening. Four stems of ‘Best Gold’, either containing female flowers or no female flowers, were attained at horticultural harvest-maturity, and were monitored for changes in colour in a vase-life room for two weeks (refer Section 4.2.1.1).

Some leaves of ‘Best Gold’ naturally contain regions of pigmentation, of the same yellow-gold colour as occurs in the spathe. This divergence in leaf morphology was not observed in Z. aethiopica grown in the gardens of Massey University and,

therefore, in the current experiment only yellow-pigmented leaves of ‘Best Gold’ were studied for changes in colour over two weeks after the leaves had unfurled.

4.2.2

Application of cytokinin

As described in Chapter 3, discs excised from the spathe of ‘Best Gold’ followed a similar pattern of re-greening as that occurring in the detached entire spathe. Hence in the current study, discs of spathe tissue were used for evaluating the effect of cytokinin in re-greening for both Z. aethiopica and ‘Best Gold’ sampled at

horticultural harvest-maturity. Using a cork borer (diameter 14 mm), discs of spathe tissue were sampled from either side of the midrib at basal, central and upper positions of spathes for both Z. aethiopica and ‘Best Gold’. The discs were placed on

filter paper soaked in treatment solutions with the adaxial surface facing down in closed petri dishes.

Pais and Neves (1982-1983) reported that application of the cytokinin-like compound 6-(o-hydroxybenzylamino)-purine at 10-4 M stimulated re-greening in the spathe of Z. aethiopica with the spadix removed. 6-benzylaminopurine (BAP) is a

synthetic cytokinin that is readily available, close in structure to the compound used in the study conducted by Pais and Neves (1982-1983), and physiologically active in

Zantedeschia (Subbaraj et al., 2010). Hence, in the current study, either BAP at 10-4

M or RO water (the control) was used as treatment solutions.

From the time of harvest the colour of the abaxial surface of discs was monitored, throughout a 14- to 17-day period of observation. Colour measurement of individual discs was discontinued early if tissue senesced, illustrated by visible browning. There were four individual stem replicates per treatment and two discs per position per spathe.

4.2.3

Pigment analysis

Discs from the central position of spathe of Z. aethiopica and ‘Best Gold’,

sampled either at horticultural harvest-maturity (day 0) or when re-greened (day 14), were analysed for pigment content. There were three stem replicates and four discs per replicate. Pigment extraction and quantification was conducted as described in Chapter 3, which comprised the freeze-dried tissue first being extracted in acetone: methanol (7:3; v:v). The supernatant was combined and dried under a stream of O2- free N2. The extract was then resuspended in ethyl acetate and kept at -20 ºC pending pigment identification and quantification by high performance liquid chromatography.

4.2.4

Statistical analysis

Data were tested initially to ensure they met the requirement for ANOVA using the general linear procedure of SAS (SAS 9.1; SAS Institute, Cary, NC). Where significant (P < 0.05) treatment effects were detected, means were separated by using

the unrestricted LSD procedure.