Despite major advances in forest biotechnology, clonal regeneration by somatic embryogenesis or organogenesis is still difficult for many woody species and is often limited to the use of juvenile explants. Adventitious regeneration of plants from gymnosperms older than zygotic embryos, and frequently even from highly immature zygotic embryos, is often difficult or has not yet been achieved. A number of experimental approaches that could eventually lead to overcoming recalcitrance are suggested in this review. When cloning trees of various ages, it is important to determine first which part of the individual contains the most responsive cells and at what time of the year these cells are in the most responsive state. This allows selection of the most useful explants. In hardwood trees and a few gymnosperms, responsive tissues are found in root or stump sprouts and in tissues near the site of meiosis at about the time that meiosis takes place. Another potentially active area is the shoot apex with most or all of its leaf or needle primordia removed. Apomixis is a natural form of clonal regeneration but occurs naturally in only one gymnosperm species. As the genetic mechanism of apomixis has been in part elucidated, the induction of apomixis by experimental means may soon be possible. The cytoplasm plays a major role in the expression or repression of nuclear genes that control embryogenesis. Expression of nuclear genes can be manipulated by nuclear transfer into de-nucleated cells (e.g., the cytoplasm of egg cells). Cytoplasmic control also plays a role in regeneration by androgenesis, asymmetric cell division and cell isolation. A short overview is presented of the genetic mechanisms involved in embryo initiation, maturation and germination and how manipulation of these mechanisms by genetic transformation could help in overcoming recalcitrance. It is expected that rapid development in the fields of research areas discussed in this review will over time eliminate the problem of recalcitrance in many instances where it is currently prevalent.

Shoot and embryolike structures were obtained in cultures of various tissues of mature conifer trees. Morphogenesis occurred in haploid and diploid tissues of female cones of Pinus mugo and Picea abies, although only rarely. Vegetative shoots dissected from early spring buds of Abies balsamea, Picea glauca, and Pseudotsuga menziesii were more responsive, with most of the organized growth occurring at the base of young needles. The following sequence of treatments was most effective: (a) collect twigs with buds shortly before budbreak, (b) cold store for at least 1 month, (c) force buds to break before excision of the vegetative shoots (keep submerged in water during excision), and (d) soak explant in malonic acid, 1 g/l, for 15 min before transfer of the shoots to the nutrient medium. ? 1981 Tissue Culture Association, Inc.

Shoot and embryolike structures were obtained in cultures of various tissues of mature conifer trees. Morphogenesis occurred in haploid and diploid tissues of female cones of Pinus mugo and Picea abies, although only rarely. Vegetative shoots dissected from early spring buds of Abies balsamea, Picea glauca, and Pseudotsuga menziesii were more responsive, with most of the organized growth occurring at the base of young needles. The following sequence of treatments was most effective: (a) collect twigs with buds shortly before budbreak, (b) cold store for at least 1 month, (c) force buds to break before excision of the vegetative shoots (keep submerged in water during excision), and (d) soak explant in malonic acid, 1 g/l, for 15 min before transfer of the shoots to the nutrient medium. ? 1981 Tissue Culture Association, Inc.