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

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 expiants 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 expiants. 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.

Temporal Competence of Embryonic Pinus Ponderosa Cotyledons to Form Multiple Buds in Vitro

“…With Picea abies, the capacity for bud production decreased ifembryos were not exposed to BA within 48 hr (von Arnold and Eriksson, 1985). In Pinus strobus, a sharp decline in organogenic competence occurred in the cotyledons within 2 days, yet 14 days were required for total loss ofcompetence (Flinn, 1987). Furthermore, Villalobos, Leung, and Thorpe (1984b) determined that light, as well as cytokinin, played a role in bud induction.…”

“…With Pinus radiata (Biondi and Thorpe, 1982), optimal bud production was achieved only after a 3-week exposure to BA. Pinus strobus (Flinn, 1987), Pinus taeda (Mott and Amerson, 1981) and Douglas fir buds (Thompson and Zaerr, 1981) required a 1,2, and 2-to-3 week BA exposure, respectively, for maximum bud induction. David (1982) reported that a concentration as high as 2.5 x 10-5 M (55 mg/l) BA for 45 days might be necessary for bud induction, depending on the anticipated morphogenic response and the degree of reactivity of the utilized explant.…”

“…1). Prior to this study, investigations of organogenic competence have used isolated cotyledons (Cheah and Cheng, 1978;Yeung et al, 1981;Patel and Thorpe, 1984;Aitken-Christie et al, 1985;Flinn, 1987). By using intact embryos, the response ofgenotypically identical cotyledons to an organogenic stimulus can be compared without concern for wound responses generated by excision or environmental differences (regardless of how small) that may be inherent among tissue culture vials.…”

Biochemical events during germination and early growth of somatic embryos and seed of interior spruce (Picea glauca engelmannicomplex)

“…1). Lipids, primarily triglycerides, are the predominant reserve substance of conifer seeds, accounting for up to 50% of cross-sectional area (Flinn et al, 1989). Feirer et al (1989) observed that somatic embryos of Norway spruce (Picea abies) contain fewer lipid bodies and lower triglyceride levels than seed embryos.…”

“…Embryogenic cultures of interior spruce were obtained from immature embryos of two open-pollinated families of interior spruce (East Kootenay [EK10], Prince George [PG118]) as outlined by Webb et al (1989). Through application of molecular probes, the maternal parentages of the populations have been identified as P. engelmannii and as P. glauca, respectively (Sutton et al, in press).…”