Morphometric analysis of reserve substances and ultrastructural changes during caulogenic determination and loss of competence of Eastern White pine (Pinus strobus) cotyledons in vitro

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Abstract
Lipid, starch, and protein reserves were monitored cytochemically and, along with certain ultrastructural changes, were quantified morphometrically during adventitious shoot determination or loss of competence in cotyledons from cultured Pinus strobus zygotic embryos. Initial explants were rich in lipid, which declined during culture. There was more lipid retention in explants on cytokinin medium by day 5 than on basal medium. However, by day 7, five- and six-celled clusters showed greater lipid utilization than most other cells on cytokinin, which may have indicated a shift towards shoot determination in these cells. Except for an initially greater retention on cytokinin, a similar pattern for storage protein degradation was observed in both treatments. Starch levels increased during culture, but did not differ between treatments during the first 7 days. Vacuolation increased during culture and was greater on basal medium than on cytokinin. Relative nuclear size also increased, but was greater on cytokinin. Relative mitochondrial area increased during culture, except in noncluster cells on basal medium. While few differences were noted between cell types within each treatment, distinct differences in nuclear size, vacuolation, and lipid content existed between cells on cytokinin and basal medium. These coincided with the timing of caulogenic determination in cytokinin-treated explants. The loss of lipid reserves, as well as ultrastructural changes associated with the maturation of cells on basal medium, was associated with the loss of competence.

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