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  • Author or Editor: John M. Sherman x
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An efficient, high-frequency regeneration and Agrobacterium-mediated transformation system was developed allowing the genetic engineering of three chrysanthemum (Dendranthema grandiflora Tzvelev) cultivars: the formerly recalcitrant and economically important cut-flower mum `Polaris' and two potted mums, `Hekla' and `Iridon'. The regeneration protocol used leaf explants on a sequence of media with four hormone regimes. Explants were first cultured on an embryogenesis-type medium containing a high concentration of 2,4-D, which promoted callus formation. Shoot primordia were induced by culture on medium lacking 2,4-D, followed by shoot elongation on a high-cytokinin plus gibberellic acid medium. Finally, elongated shoots were rooted on a low-auxin rooting medium. Transformed plants of the three cultivars were obtained following co-culture of leaf explants with A. tumefaciens strain EHA 105 harboring the plasmid pBI121 containing genes for neomycin phosphotransferase II (NPTII) and β-glucuronidase (GUS). Stable transformation of the three cultivars was verified via GUS assays and Southern analysis.

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Poncirus trifoliata (L.) Raf. seeds were germinated in perlite under intermittent mist at about 25 °C and natural daylight in a greenhouse. Two-week-old seedlings were then transferred into a growth chamber at 25 °C and 16-hour daylength for 1 week. Tissue samples were collected at 0, 6, 24, 168, and 504 hours after temperature equilibration at 10 °C. Freezing tolerance at –6.7 °C, as determined by electrolyte leakage, and stem (leaves attached) water potential (ψx), measured using a pressure chamber, was recorded for a subset of seedlings for each time interval. Red coloration (apparently anthocyanin) developed at the petiole leaflet junction and buds after 48 hours at 10 °C and gradually occurred throughout the leaves during further exposure. Complementary DNA clones for phenylalanine ammonia lyase (PAL), 4-coumarate: coA ligase (4CL), and chalcone synthase (CHS) were used to probe RNA isolated from the leaves. No increase in steady-state messenger RNA level was detected. Increases in freeze hardiness occurred within 6 hours in the leaves, and continued for up to 1 week. Water potential initially decreased from –0.6 to –2.0 MPa after 6 hours, then returned to –0.6 MPa after 1 week. Thus, Poncirus trifoliata seedlings freeze-acclimate significantly after only 6 hours at 10 °C.

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Poncirus trifoliata is a comparatively hardy, cross compatible, and graft compliant relative of Citrus. The citrus industry in Florida has suffered immense economic losses due to freezes. Although much research has been done in citrus freeze hardiness, little work has been on the early induction of freeze tolerance by low temperature. Poncirus trifoliata `Rubidoux' seedlings were germinated in perlite under intermittent mist at about 25°C and natural daylight conditions in a greenhouse and grown 2 weeks. See dlings were then transferred into a growth chamber at 25°C and 16 hour daylength for 1 week. Temperature was lowered to 10°C and tissue samples were collected at 0, 6, 24, and 168 hours. Freezing tolerance, at –6.7°C as determined by electrolyte leakage, and stem (leaves attached) water potential, measured using a pressure bomb, were also recorded for a subset of seedlings for the above intervals. After exposure to low temperature for 48 hours a red coloration became visible at the petiole leaflet junction an d at the buds, with subsequent exposure to low temperature the coloration spread to the leaves. Clones for phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL), and chlorophyll ab binding protein (CAB), and chalcone synthase (CHS) were used to probe RNA isolated from P. trifoliata. PAL and 4CL transcripts increased in response to the low temperature. Significant increases in freeze hardiness occurred within 6 hours in the leaves, and increases continued for up to one week. Water potential increased from –0.6 to –2.0 MPa after 6 hours, then returned to –0.6 MPa after 1 week. These data indicate that increases in freezing tolerance and changes in water potential and gene expression can be detected shortly after low temperature treatments are imposed on P. trifoliata seedlings.

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