Embryogenic cultures of Daucus carota treated with 1 millimolar alpha-difluoromethylarginine, a specific inhibitor of arginine decarboxylase, exhibited nearly a 50 percent reduction in embryo formation compared with controls. Putrescine and spermidine concentrations in the treated cells were greatly reduced. Addition of putrescine, spermidine, or spermine to the culture medium restored embryogenesis in the treated cultures. Embryogenesis was not significantly affected by alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase. These results suggest that polyamines have a major function in plant embryo development and that the wild carrot synthesizes polyamines through the biosynthetic pathway involving arginine decarboxylase rather than ornithine decarboxylase.
Our progress is reviewed on development of somatic embryogenesis in conifers for mass propagation. A distinct embryogenic callus (EC) phenotype, white, mucilaginous, and rapidly growing, has been initiated on modified MS media with 2,4-D or NAA (2-5 mg/L) and BA(0-1 mg/L) from immature embryos of Norway spruce (Picea abies), white spruce (Picea glauca), loblolly pine (Pinus taeda), pond pine (Pinus serotina), and white pine (Pinus strobus). EC has also been initiated from mature embryos of Norway spruce and maintained as rapidly growing (48 hour doubling) liquid suspensions. Initiation of EC in Picea and Pinus differ markedly in several ways. Precotyledonary embryos were optimal in Pinus and EC originated from the suspensor region. In Picea EC originated from the hypocotyl and cotyledon region of predominantly post-cotyledonary embryos. Biochemically, EC of Picea and Pinus were similar and distinctly different from nonembryogenic callus (NEC) in terms of ethylene evolution rates (EC low and NEC high), level of total reductants, including glutathione (EC low and NEC high), and protein synthesis rates (EC high and NEC low). Conifer somatic embryos contained proplastids closely resembling those found in early zygotic embryos. On proliferation medium in the light, EC was white and maintained the proplastid morphology, whereas, NEC was green and contained mature chloroplasts with grana. These biochemical and ultrastructural differences served to both verify and predict embryogenic potential.With Norway spruce somatic embryos, maturation frequencies as high as 25% have been attained. Germination frequencies as high as 82% (mean 56%) have been obtained. Twenty-nine percent of the somatic embryo plantlets survived transfer to the greenhouse, set a dormant terminal bud, overwintered to -5°C, and renewed vegetative growth synchronously with control seedlings. This is the first report of overwintering and renewed vegetative growth from resting buds of conifer somatic embryo plants.
Triglycerides in developing zygotic embryos of Norway spruce and loblolly pine were found to accumulate continuously during the course of development, comprising nearly 50% of the fresh weight of a mature embryo. Embryogenic calli of these two species contained dramatically lower levels of triglycerides. Abscisic acid treatments promoted both embryo production and triglyceride accumulation in Norway spruce cultures. A method used to determine triglyceride levels in human serum, commercially available in kit form, was adapted for use with plant tissues. Low levels of triglycerides in the cultured tissues may be related to difficulties in the development and germination of conifer somatic embryos.
The spermidine synthesis inhibitors methylglyoxal bis-(guanylhydrazone) (MGBG) and dicyclohexylammordum sulfate (DCHA) were found to reduce growth and embryogenesis in wild carrot cultures. Cellular polyamine levels were also affected by the inhibitors, with spermidine levels being especially reduced by DCHA. Similarly, MGBG reduced organogenetic development of shoots on excised aspen hypocotyls. These data suggest that the polyamines, especially spermidine, play an important role in the growth and development of plants.
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