P. von Aderkas

Recalcitrance in clonal propagation, in particular of conifers

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.

Rejuvenation of Tissues from Mature Conifers and its Implications for Propagation in Vitro

Clonal propagation of trees old enough to have expressed their desirable characteristics is an effective means of rapidly obtaining improved planting stock. However, cloning of mature trees of conifer species is often difficult or impossible with present-day techniques. Therefore, specimens are often selected for clonal propagation when they are still too young for proper assesment. For many species such assesment is, at present, not possible until the trees have reached about half their rotation age (Namkoong et al. 1980). Because of this predicament, maximum benefit of clonal propagation cannot be expected until we find the appropriate means either to clone older trees or to determine at a young age what the trees will be like at the end of their rotation.

In vitro fertilization from co-cultured pollen tubes and female gametophytes of Douglas fir (Pseudotsuga menziesii)

Abstract Our previous attempt on in vitro fertilization (IVF) in conifers resulted in pollen tube penetration of female gametophytes, but because of the rapid decline in egg viability, no further interaction occurred. In this report, we describe for the first time that IVF has been achieved in conifers. Using Douglas fir (Pseudotsuga menziesii), we describe a two-step process which involved induction of pollen tubes in culture followed by introduction of isolated female gametophytes at the tips of growing pollen tubes. Pollen tubes penetrated the introduced isolated female gametophytes at various places, but a number of tubes entered the egg cell through the neck cells similar to the in vivo condition. Under our current culture conditions, longevity of pollen tubes and eggs has been improved resulting in the release of sperms, fusion of gametes, and initial formation of the proembryo. Continued plasmolysis of the egg limited the number of successful gametic interactions. IVF has been accomplished in flowering plants in several ways, but the gametophyte-gametophyte IVF system described in this paper is unique. IVF offers a novel breeding technology that takes advantage of the sexual reproductive route. When coupled with hybridization and genetic transformation, IVF could result in the development of stable novel genotypes of economically superior trees.

Embryogenesis and genetic stability in long term megagametophyte-derived cultures of larch

Embryogenic cultures derived from megagametophytes of Larix decidua were maintained for up to 17 years. A few lines were divided into sub-lines, which were maintained in the same manner as the others. Embryogenic tissue was grown on 1/2 strength LM medium supplemented with glutamine and casein hydrolysate at constant temperature and light regimes. Chromosome counts were conducted at various times. DNA content was assessed by flow cytometry. Embryogenesis was monitored with each transfer and records of all appearances of green mature embryos were kept. Chromosome number was found to vary. DNA content and chromosome number, both of which had doubled a number of years after initiation, stabilized around 24 chromosomes for most cultures. A few lines showed substantial increases in chromosome number. One of these lines lost vigour and died. Another line showed a further doubling of DNA content. No lines were embryogenic over the entire period. Embryogenicity was lost completely in some lines, but in others the loss was temporary, as periodic restoration of embryogenesis was observed.

Micropylar exudates in Douglas fir – timing and volume of production

Abstract In Pseudotsuga menziesii, a secretion fills the micropylar canal about 7 weeks postpollination until fertilization. Micropylar volumes were measured and found to show variation. Dissection of the ovuliferous scales caused excess fluid to be exuded from the micropylar canal, forming a drop at the tip of the micropyle. This drop was collected, and its production quantified in three trees. Volume and percentage of ovules with drops were greatest when the archegonia in the female gametophyte were at central cell and/or egg cell stage. The volume of exuded drops far exceeded that of the micropyle. Production of subsequent drops by the ovules further confirms that the fluid is actively produced upon dissection

Effects of endogenous ABA levels and temperature on cedar (Cedrus libani Loudon) bud dormancy in vitro

Abstract Axillary and apical buds of in-vitro-propagated cuttings of Cedrus libani are unable to burst at 24 °C, but this inhibition was overcome at 30 °C. Here we have used cedar microcuttings to investigate whether the levels of endogenous hormones vary with bud dormancy and temperature. We analysed the levels of abscisic acid, indole-3-acetic acid, zeatin, isopentenyladenine and their major metabolites using HPLC purification and fractionation of the samples coupled to an ELISA method for hormonal quantitation involving several antibodies elicited against each hormonal family. Abscisic acid levels in microcuttings with dormant buds were higher than those in microcuttings with growing buds. At 24 °C, needles accumulated more abscisic acid than at 30 °C. In addition, when needles were removed, but growth release was achieved at 24 °C. Abscisic acid supplied at 30 °C induced the formation of dormant buds. These results suggest that abscisic acid accumulation in the needles can explain the bud dormancy of cedar microcuttings at 24 °C.

Plants from haploid tissue culture of Lavix decidua.

Haploid embryogenic tissue was initiated on 1/2 LM medium supplemented with 500 mg/l glutamine, 1000 mg/l casein hydrolysate, 100 mg/l inositol, 30000 mg/l sucrose, and 0.1 mg/l 2,4-D. The embryoids matured to produce plantlets. One plant from one of the two lines survived. The chromosome complement of tissue cultures, of the needle bases from the source plant, and of the plant produced in vitro were established by squashes. DNA content was assessed by DNA microdensiometry. In vitro tissues were haploid (n = 12). The plant produced was mixoploid, with a predominance of diploid cells (2n = 24).

Intergeneric pollen : megagametophyte relationships of conifers in vitro

Abstract Germinating pollen from larch (Larix occidentalis), Sitka spruce (Picea sitchensis) and white pine (Pinus monticola) were co-cultured with megagametophytes dissected from cones of other genera (Pseudotsuga menziesii, Larix×eurolepis and Pinus monticola). Pollen was presented to megagametophytes possessing archegonia which were either alive, degenerating or dead. In addition, pollen was presented to fertilized megagametophytes and to megagametophytes that had been cut in half. Megagametophyte penetration by pollen tubes and male gamete release into archegonia were verified by serial sections of glycomethacrylate-embedded specimens. Pollen tubes penetrated through any part of the apex of the megagametophyte. Division of the body cell into the two gametes was regularly observed. Delivery of gametes was confirmed between spruce and larch. Pollen tubes also penetrated fertilized megagametophytes, dead or degenerating archegonia as well as wounded and/or cut surfaces. This demonstrates the inability of the male gametophyte to optimize its mating efforts, since it is unable to differentiate between healthy and unhealthy archegonia. The megagametophyte cells are unable to optimize male selection. They may produce secretions of a generally attractive nature, as pollen is attracted to the apex of the megagametophyte, but archegonia themselves do not produce pollen-specific signals of either a promotive or inhibitory nature. These results open new avenues for the development of novel breeding strategies where natural breeding barriers may be bypassed.

Diploidization in megagametophyte-derived cultures of the gymnosperm Larix decidua.

Tested haploid embryogenic lines (n=12) of Larix dedicua Mill, initiated from megagametophyte tissue were maintained on half-strength LM medium without growth regulators. The cultures were analyzed for ploidy level after 1–9 years. All lines tested were found to have doubled (2n=24) their chromosome number at the end of the experiment, though there were a few lines that still gave occasional haploid counts. Flow cytometric data of embryogenic tissue confirmed these results. Protoplasts were stained in ethidium bromide, and cultured human leucocytes and chicken erythrocytes were used as internal standards. Haploid megagametophytes from immature seeds of L. decidua and known diploid culture lines of a related hybrid (L. x eurolepis) were also analyzed by flow cytometry. Haploid reference material had 12.3–13.6 pg DNA per cell, whereas formerly haploid callus lines had an average of 25.0 pg DNA per cell. The one exception was a known, genetically unstable line of L. decidua (34.8 pg DNA per cell). The diploid cell line of L. x eurolepis had 27.6 pg DNA per cell. The results show that spontaneous diploidization of megagametophyte lines is relatively rapid and that both haploid and dihaploid lines are embryogenic in larch.

Haploid embryogenesis in trees

Plant breeders have long sought haploid and double-haploid plants [22]. Haploids have been produced in numerous genera, most of them agricultural crops. Because haploid plants do not exhibit phenomena such as dominance and segregation they reveal cryptic and recessive genetic variation. Such plants are useful for producing homozygous diploid (dihaploid) lines for controlled hybridization. Traditionally, homozygosity for certain traits was produced by continuous inbreeding, but for trees this is somewhat problematic. This review concentrates on tree species of conifers (including Taxaceae) as well as angiosperms. Breeding of tree species requires longer periods of time and thus inbreeding is impractical, and in certain cases impossible. For example, inbreeding in dioecious trees is restricted to sib-mating, never selfing. Although haploids could be used to speed up bredeing programs, to study inheritance of qualitative traits and to establish linkage maps, there are relatively few tree species which have been amenable to induction of haploids.