Zygotic and somatic embryo morphogenesis in Pinus pinaster: comparative histological and histochemical study.

We compared morphogenesis and accumulation of storage proteins and starch in Pinus pinaster Ait. zygotic embryos with those in somatic embryos grown with different carbohydrate sources. The maturation medium for somatic embryos included 80 microM abscisic acid (ABA), 9 g l(-1) gellam gum and either glucose, sucrose or maltose at 44, 88, 175 or 263 mM in the presence or absence of 6% (w/v) polyethylene glycol (PEG) 4000 MW. Maturation medium containing 44 or 88 mM of a carbohydrate source produced only one or no cotyledonary somatic embryos per 0.6 g fresh mass of culture. The addition of PEG to the basal maturation medium resulted in a low yield of cotyledonary somatic embryos that generally showed incomplete development and anatomical abnormalities such as large intercellular spaces and large vacuoles. High concentrations of maltose also induced large intercellular spaces in the somatic embryonic cells, and 263 mM sucrose produced fewer and less developed cotyledonary somatic embryos compared with 175 mM sucrose, indicating that the effect of carbohydrate source is partially osmotic. Zygotic embryos had a lower dry mass than somatic embryos at the same stage of development. Starch granules followed a similar accumulation pattern in zygotic and somatic embryos. A low starch content was found in cotyledonary zygotic embryos and in somatic embryos developed in the presence of 175 mM maltose or 263 mM glucose. In zygotic embryos and in PEG-treated somatic embryos, protein bodies appeared later and were smaller and fewer than in well-developed somatic embryos grown without PEG. We propose that storage protein concentration might be a marker of embryo quality.

Cellular Location of Prune dwarf virus in Almond Sections by In Situ Reverse Transcription-Polymerase Chain Reaction.

ABSTRACT In situ reverse transcription-polymerase chain reaction (RT-PCR) was used in young leaves (from trees and in vitro shoots) and flower buds of almond (Prunus dulcis), a stone fruit, for cellular location of Prune dwarf virus (PDV, a member of the genus Ilarvirus). Sections obtained from samples fixed in formaldehyde and embedded in paraffin were refixed in formaldehyde to increase tissue preservation in the RT-PCR steps. The coat protein gene of PDV was used as the target to produce a cDNA copy that was amplified by PCR and visualized using a direct detection method with digoxigenin-labeled nucleotides. Protein digestion, PCR, and detection strategies were optimized for increased tissue preservation and signal intensity. PDV was found in infected samples within the vascular tissue of young leaves and flower buds as well as in the mesophyll in developing floral organs and in the generative and vegetative cells of pollen grains. PDV signals were observed in a ring surrounding the nucleus and spread in the cytoplasm. The results obtained are discussed in terms of the technique optimization and PDV distribution in tissues and transmission through pollen. The optimized protocol of in situ RT-PCR is a powerful technique to reveal low-abundant RNA species. Therefore, it is appropriate to study cell and subcellular distribution of RNA viruses in woody species.