Development of somatic embryos in Norway spruce

Embryogenic cell lines of Norway spruce consist of a large number of somatic embryos. The cell lines have been divided into two groups, A and B. The group B embryos are developmentally blocked. Extracts of mature spruce seeds stimulate group B embryos to develop a morphology comparable to group A embryos. However, seed extract inhibits early embryo development. The active components in seed extract were shown probably to be proteins. Extracts of mature seeds contain chitinase-like proteins as recognized by an antibody towards chitinase 4 in sugar beet. Proteins of similar sizes were detected by the same antibody in the conditioned medium of group A, but not in group B. A chitinase 4-related chitinase and a nod factor had a stimulating effect on early embryo development, but did not influence the later stages of embryo development.Key words: Picea abies, somatic embryogenesis, embryo development, seed extract.      

A key developmental switch during Norway spruce somatic embryogenesis is induced by withdrawal of growth regulators and is associated with cell death and extracellular acidification.

The biotechnology of somatic embryogenesis holds considerable promise for clonal propagation and breeding programs in forestry. To efficiently regulate the whole process of plant regeneration through somatic embryogenesis, it is of outmost importance to understand early developmental events when somatic embryos are just formed. In Norway spruce, somatic embryos transdifferentiate from proembryogenic masses (PEMs). This work describes the developmental dynamics (frequency distribution of PEMs and early somatic embryos) of the whole embryogenic suspension culture growing in the presence and absence of plant growth regulators (PGRs), auxin and cytokinin. The experiments have shown that PEM-to-somatic embryo transition is a key developmental switch that determines the yield and quality of mature somatic embryos and ultimately plant production. This switch was induced by the withdrawal of PGRs in cell suspension leading to a rapid accumulation of early somatic embryos (to a maximum of 75% of the entire population of suspension culture) and concomitant degradation of PEMs. The latter was evident from increased level of cell death measured through spectrophotometric Evans blue staining assay. Proembryogenic mass-to-embryo transition and concomitant activation of cell death were mediated by strong extracellular acidification. Therefore, buffering PGR-free culture medium at high (pH 5.8) or low (pH 4.5) levels of pH inhibited both PEM-to-embryo transition and cell death. The yield of mature somatic embryos on abscisic acid (ABA)-containing medium was increased up to 10-fold if the suspension culture had been pretreated for 1 to 9 days in unbuffered PGR-free medium. In this case a large proportion (75%) of the total number of mature embryos was formed within a short, 5-week, contact with ABA. The latter is practically important because prolonged contact with ABA suppresses the growth of somatic embryo plants. Based on these results, an improved method for regulating somatic embryogenesis was set up and tested for nine genotypes of Norway spruce. Over 800 plants regenerated from all tested genotypes demonstrated a good performance in the greenhouse and they were transferred to the field.     

Improved and synchronized maturation of Norway spruce (<Emphasis Type="Italic">Picea abies</Emphasis> (L.) H.Karst.) somatic embryos in temporary immersion bioreactors

Somatic embryogenesis offers many benefits for clonal propagation in large-scale plant production of conifers. A key rate-limiting step is the conversion from early-stage somatic embryos in pro-embryogenic masses (PEMs) to the maturation stage. Immature embryos in PEMs are present at different developmental stages, where some are unable to respond to the maturation treatment, thus limiting yields of mature embryos. Synchronization of early somatic embryo development in PEMs could greatly improve subsequent yields of mature embryos. A temporary immersion bioreactor designed for Norway spruce (Picea abies (L.) H.Karst.) was used in this study. Through a specific system for dispersion, connected tissue of PEMs, composed of immature embryos grown in liquid medium in the temporary immersion bioreactors or on solid medium as a control, was dispersed and redistributed in a more uniform spatial arrangement. It was demonstrated that development of mature embryos could be significantly stimulated by dispersion, compared to controls, in both medium types. Synchronization of maturation was evaluated by a statistical approach. The present study shows that the yield of mature embryos from dispersed PEMs was three to five times higher than that from non-dispersed controls in three of four cell lines of Norway spruce tested, both in bioreactors and on solid medium.     

Propagation of Norway spruce via somatic embryogenesis

Somatic embryogenesis combined with cryopreservation is an attractive method to propagate Norway spruce (Picea abies) vegetatively both as a tool in the breeding programme and for large-scale clonal propagation of elite material. Somatic embryos are also a valuable tool for studying regulation of embryo development. Embryogenic cell lines of Norway spruce are established from zygotic embryos. The cell lines proliferate as proembryogenic masses (PEMs). Somatic embryos develop from PEMs. PEM-to-somatic embryo transition is a key developmental switch that determines the yield and quality of mature somatic embryos. Withdrawal of plant growth regulators (PGRs) stimulates PEM-to-somatic embryo transition accompanied by programmed cell death (PCD) in PEMs. This PCD is mediated by a marked decrease in extracellular pH. If the acidification is abolished by buffering the culture medium, PEM-to-somatic embryo transition together with PCD is inhibited. Cell death, induced by withdrawal of PGRs, can be suppressed by extra supply of lipo-chitooligosaccharides (LCOs). Extracellular chitinases are probably involved in production and degradation of LCOs. During early embryogeny, the embryos form an embryonal mass surrounded by a surface layer. The formation of a surface layer is accompanied by a switch in the expression pattern of an Ltp-like gene (Pa18) and a homeobox gene (PaHB1), from ubiquitous expression in PEMs to surface layer-specific in somatic embryos. Ectopic expression of Pa18 and PaHB1 leads to an early developmental block. Transgenic embryos and plants of Norway spruce are routinely produced by using a biolistic approach. The transgenic material is used for studying the importance of specific genes for regulating plant development, but transgenic plants can also be used for identification of candidate genes for use in the breeding programme.     

Inhibited polar auxin transport results in aberrant embryo development in Norway spruce

Current hypotheses concerning the role of polar auxin transport in embryo development are entirely based on studies of angiosperms, while little is known about how auxin regulates pattern formation in gymnosperms. In this study, different developmental stages of somatic embryos of Norway spruce (Picea abies) were treated with the polar auxin transport inhibitor 1-N-naphtylphthalamic acid (NPA). Effects of the treatments on auxin content, embryo differentiation and programmed cell death (PCD) were analysed. During early embryo development, NPA-treatment led to increased indole-3-acetic acid (IAA) content, abnormal cell divisions and decreased PCD, resulting in aberrant development of embryonal tube cells and suspensors. Mature embryos that had been treated with NPA showed both apical and basal abnormalities. Typically the embryos had abnormal cotyledon formation and irregular cell divisions in the area of the root meristem. Our results show that polar auxin transport is essential for the correct patterning of both apical and basal parts of conifer embryos throughout the whole developmental process. Furthermore, the aberrant morhologies of NPA-treated spruce embryos are comparable with several auxin response and transport mutants in Arabidopsis. This suggests that the role of polar auxin transport is conserved between angiosperms and gymnosperms.     

High-efficiency somatic embryogenesis and plant regeneration from suspension cultures of grapevine

Embryogenic suspensions of grapevine (Vitis vinifera L.) were initiated from somatic embryos of `Thompson Seedless' and `Chardonnay'. Suspension cultures consisted of proembryonic masses (PEM) that proliferated without differentiation in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). `Chardonnay' somatic embryos developed fully from PEMs following subculture in medium without 2,4-D; however, somatic embryo development did not advance beyond the heart stage in `Thompson Seedless' suspension cultures. Highly synchronized development of somatic embryos was obtained by inoculating <960-μm PEMs into liquid medium without 2,4-D. Somatic embryos were also produced in large numbers from suspension-derived PEMs of both cultivars on semisolid medium lacking 2,4-D. Somatic embryos matured and regenerated into plants in MS basal medium containing 3% sucrose. Using this method more than 60% of the somatic embryos regenerated plants. More than 90% of the regenerated plants were successfully transferred to the greenhouse. Received: 27 July 1998 / Revision received: 15 October 1998 / Accepted: 27 October 1998     

Polar auxin transport controls suspensor fate

Polar auxin transport is critical for normal embryo development in angiosperms. It has been proposed that auxin accumulates dynamically at specific positions, which in early Arabidopsis embryos correlates with developmental decisions such as specification of the apical cell lineage, specification of the hypophysis, and differentiation of the two cotyledons. In conifers, pattern formation during embryo development is different, and includes a free nuclear stage, nondividing suspensor cells, presence of tube cells, lack of hypophysis and formation of a crown of cotyledons surrounding the shoot apical meristem. We have recently shown that polar auxin transport is important for normal embryo development also in conifers. Here we suggest a model where auxin is transported from the suspensor cells to the embryonal mass during early embryogeny in conifers. This transport is essential for the developmental decisions of the tube cells and the suspensor, and affects both the amount of programmed cell death and the embryo patterning.Key words: conifer, embryo development, 1-N-naphtylphthalamic acid (NPA), patterning, polar auxin transport, programmed cell death, somatic embryogenesis, suspensorIn the model plant Arabidopsis thaliana auxin is transported, already from the first cell division of the zygote, from the basal cell to the apical cell, where it is involved in establishing the identity of the apical cell lineage. At the 32-cell stage the polar auxin transport is reversed, leading to an auxin accumulation in the uppermost suspensor cell, which occurs concomitantly with the specification of the hypophysis. During the heart stage auxin is transported towards the cotyledonary primordia, giving positional information about the cotyledon outgrowth.¹ Formation of the apical-basal embryonic pattern during early embryogeny in conifers is quite different from that in Arabidopsis and proceeds through the establishment of three major cell types: the meristematic cells of the embryonal mass, the embryonal tube cells and terminally differentiated nondividing suspensor cells.²The somatic embryo system of Picea abies (Norway spruce) includes a stereotyped sequence of developmental stages, resembling zygotic embryogeny, which can be synchronized by specific treatments.^3,4 We are using this as a model system for elucidating the regulation of embryo development in conifers.² Early somatic embryos differentiate from proembryonic masses (PEMs) after withdrawal of the plant growth regulators (PGRs) auxin and cytokinin (Fig. 1A and B). We have previously shown that the organisation of the apical-basal polarity in early embryos is dependent on a gradient of PCD from the embryonal tube cells committed to death, to the cell corpses at the basal end of the suspensor.^5–7 Dysregulation of the PCD leads to aberrant apical-basal patterning.Open in a separate windowFigure 1Model for polar auxin transport control of early embryo patterning in conifers. (A) Embryogenic cultures proliferate as proembryonic masses (PEMs) in the presence of the plant growth regulators (PGRs) auxin and cytokinin. (B) Early embryos start to differentiate from PEMs after withdrawal of PGRs. Endogenous auxin is transported to the newly formed embryonal mass. (C) Early embryos are formed within two weeks in PGR-free medium. Early embryos have a distinct embryonal mass, tube cells and a suspensor. IAA is transported from the suspensor and the tube cells to the embryonal mass. (D) Fully matured cotyledonary embryos are formed after 5–6 weeks on maturation medium. (E) Treatment with NPA blocks the polar auxin transport to the embryonal mass, leading to an IAA accumulation in the suspensor cells, tube cells and perhaps also in the cells of the embryonal mass most adjacent to the tube cells. (F) Embryos with supernumerary suspensor cells are formed if polar auxin transport is inhibited only during the earliest stages of suspensor differentiation. (G) Embryos with meristematic cells in the suspensor are formed if polar auxin transport is inhibited during both differentiation and elongation of the suspensor. We assume that these abnormalities abort further development and maturation of viable embryos. em, embryonal mass; s, suspensor; tc, tube cells. Green arrows indicate polar auxin transport, T indicates blocked polar auxin transport, green shadings indicate auxin accumulation.We recently showed that in embryogenic cultures of Norway spruce treated with the polar auxin transport inhibitor NPA, the number of cells undergoing PCD decreases. As a consequence the balance between the number of cells in the embryonal mass and the number of cells in the suspensor develop abnormally, and concomitantly the endogenous free IAA content increases almost two-fold.⁸In order to visualise the IAA accumulation within the embryos we used a -318 bp deletion of the auxin-responsive IAA4/5 promoter from Pisum sativum (pea), previously characterized by Oeller et al.,⁹ and Ballas et al.,¹⁰ fused to the GUS reporter gene.¹¹ In tobacco (Nicotiana tabacum) the promoter is expressed in rapidly elongating hypocotyls,¹² (our unpublished observations) and strong induction by auxin is clear in elongating zones of both roots and hypocotyls in transgenic pIAA4/5-GUS Arabidopsis plants.¹¹ However, to our knowledge, expression of IAA4/5 has not been reported in embryonal shoot apical meristems. Hence, the pIAA4/5-GUS may preferentially be used as a biosensor of auxin activity in non-meristematic cells during spruce embryo development. During normal somatic embryo development in spruce, pIAA4/5-GUS activity is detected in PEMs, tube cells and suspensor cells, but not in the embryonal mass. Early embryos of Norway spruce that are treated with NPA show increased pIAA4/5-GUS activity in tube cells and suspensor cells (unpublished), well in line with the increment of free IAA levels.Our results indicate that IAA under normal conditions is transported from the suspensor cells to the cells in the embryonal mass (Fig. 1B and C). NPA-treatment blocks this polar transport of endogenous IAA, which results in an accumulation of IAA and increased pIAA4/5-GUS activity in the suspensor cells, the tube cells, and perhaps also in the cells of the embryonal mass most adjacent to the tube cells (Fig. 1F and G). Blocked polar auxin transport during early differentiation of the suspensor stimulates abnormal cell divisions of the meristematic cells most adjacent to the tube cells or perhaps even of the tube cells themselves. Consequently, embryos with supernumerary tube and suspensor cells are formed (Fig. 1F). If the polar auxin transport is blocked for a longer time, i.e., during both differentiation and elongation of the suspensor, the auxin accumulation leads to maintenance of meristematic fate and a failure to undergo PCD (Fig. 1G).It has been proposed that the fate of the suspensor cells is regulated by signals from the embryo proper which impede developmental potential and initiate PCD.¹³ In accordance, we assume that the abnormal embryo morphologies formed after NPA-treatment may result from adverse inhibitory signals from the embryonal mass.     

Developmental pathway of somatic embryogenesis in Picea abies as revealed by time-lapse tracking

Several coniferous species can be propagated via somatic embryogenesis. This is a useful method for clonal propagation, but it can also be used for studying how embryo development is regulated in conifers. However, in conifers it is not known to what extent somatic and zygotic embryos develop similarly, because there has been little research on the origin and development of somatic embryos. A time-lapse tracking technique has been set up, and the development of more than 2000 single cells and few-celled aggregates isolated from embryogenic suspension cultures of Norway spruce (Picea abies L. Karst.) and embedded in thin layers of agarose has been traced. Experiments have shown that somatic embryos develop from proembryogenic masses which pass through a series of three characteristic stages distinguished by cellular organization and cell number (stages I, II and III) to transdifferentiate to somatic embryos. Microscopic inspection of different types of structures has revealed that proembryogenic masses are characterized by high interclonal variation of shape and cellular constitution. In contrast, somatic embryos are morphologically conservative structures, possessing a distinct protoderm-like cell layer as well as embryonal tube cells and suspensor. The lack of staining of the arabinogalactan protein epitope recognized by the monoclonal antibody JIM13 was shown to be an efficient marker for distinguishing proembryogenic masses from somatic embryos. The vast majority of cells in proembryogenic masses expressed this epitope and none of cells in the early somatic embryos. The conditions that promote cell proliferation (i.e. the presence of exogenous auxin and cytokinin), inhibit somatic embryo formation; instead, continuous multiplication of stage I proembryogenic masses by unequal division of embryogenic cells with dense cytoplasm is the prevailing process. Once somatic embryos have formed, their further development to mature forms requires abscisic acid and shares a common histodifferentiation pattern with zygotic embryos. Although the earliest stages of somatic embryo development comparable to proembryogeny could not be characterized, the subsequent developmental processes correspond closely to what occurs in the course of early and late zygotic embryogeny. A model for somatic embryogenesis pathways in Picea abies is presented.     

Evaluation of somaclonal variation during somatic embryogenesis of interior spruce (Picea glauca engelmannii complex) using culture morphology and isozyme analysis

Somaclonal variation during interior spruce (Picea glauca engelmannii complex) somatic embryogenesis was evaluated using culture morphology and isozyme analysis. Genotype-specific abscisic acid-dependent developmental profiles and isozyme patterns were similar for subclone and parent line embryogenic cultures and cotyledonary somatic embryos. Extensive analysis of fifteen hundred subclone embryos of one genotype revealed no isozyme pattern variation. Initiation of embryogenic cultures was dependent on the developmental stage of the explant although cultures derived from different stages were morphologically similar. The embryogenic cultures initiated from interior spruce embryos show a high degree of genetic stability in that the morphological behavior and isozyme phenotype were always consistent with that of the explant genotype. These results support the conclusion that this culture system is appropriate for clonal propagation of interior spruce.     

Overexpression of HBK3, a class I KNOX homeobox gene, improves the development of Norway spruce (Picea abies) somatic embryos

In order to investigate the effects of HBK3, a spruce gene member of the class I KNOX family, during somatic embryogenesis, sense (HBK3-S) and antisense (HBK3-A) Norway spruce (Picea abies) lines were generated. Somatic embryos produced from these lines were then analysed at morphological and structural levels. Compared with control, differentiation of immature somatic embryos from pro-embryogenic masses (PEMs) was accelerated in lines overexpressing HBK3 (HBK3-S). Such immature embryos showed enlarged embryogenic heads and were able to produce fully developed cotyledonary embryos at higher frequency. Furthermore, HBK3-S embryos had enlarged shoot apical meristems (SAMs) and enlarged expression pattern of PgAGO, a molecular marker gene specific to meristematic cells. Lines in which HBK3 (HBK3-A) was down-regulated had reduced ability to produce immature somatic embryos from PEMs and were not able to complete the maturation processes. To assess the function of HBK3 in comparison with that of angiosperm KNOX genes, this gene was ectopically expressed in Arabidopsis plants. As observed for spruce, Arabidopsis embryos overexpressing HBK3 had enlarged meristems and enlarged expression pattern of SHOOTMERISTEMLESS, a SAM molecular marker gene. In addition, transformed embryos were able to germinate at a higher rate and the resulting plants showed a variety of phenotypic aberrations, including abnormal leaves and reduced apical dominance. Overall, these data confirm the importance of KNOTTED genes during development and reveal the participation of HBK3 in conifer embryogeny. Furthermore, the results show redundant functions of this gene during embryonic growth of spruce and Arabidopsis, but not during post-embryonic growth.     

Effect of anti-auxins on maturation of embryogenic tissue cultures of Nordmanns fir (Abies nordmanniana)

The present study was conducted to improve the transition from proliferation to maturation in embryogenic cultures of Nordmanns fir. For that reason, chemicals reported to affect endogenous levels or activity of auxin were included in the growth media during maturation. The auxin antagonist PCIB reduced proliferation and promoted the development of numerous high-quality mature embryos in the tested cell lines. PCIB could not substitute for exogenously supplied ABA and the positive effect was only found when PCIB and ABA were used in combination. The effect of PCIB was dependent on the concentration and the application period. The auxin transport inhibitor TIBA also reduced proliferation, but had no positive effect on maturation. The auxin synergist phloroglucinol had the opposite effect of PCIB; proliferation was increased and no maturation was initiated. A lowered concentration of boron had no effect on proliferation but had some positive effect on maturation. The optimum protocol for PCIB application was strongly genotype dependent, and a general scheme that covered the tested cell lines could not be found. Overexposure to PCIB during maturation caused abnormal development of the mature embryos, which was revealed by a reduced number of cotyledons. These results suggest that endogenously produced auxin may be one reason for low or failing maturation of embryogenic cultures of Nordmanns fir, but also imply that auxin may play a critical role for proper development of cotyledons during the later stages of embryo maturation.     

Induction of green ash embryogenic cultures with potential for scalable somatic embryo production using suspension culture

Key message

The developmental sequences of zygotic embryos of green ash collected from the same tree were widely asynchronous and an intermediate developmental stage was the best explant for inducing somatic embryogenesis.

Abstract

All North American ash (Fraxinus) species are under threat of extirpation from their native ranges by the emerald ash borer (EAB; Agrilus planipennis), an exotic wood-boring beetle that has already destroyed millions of ash trees in 15 U.S. states and Canada. We tested treatments aimed at initiating embryogenic cultures from seeds of green ash (F. pennsylvanica), with the long-term goal of using these cultures to aid in research to generate EAB-resistant ash trees for restoration. In preparation for somatic embryogenesis induction experiments, we first defined specific stage(s) of green ash zygotic embryo development using time-tracing sampling by collecting samaras of two green ash trees from May to August in 2012. Seed development was divided into seven stages according to both seed and embryo size, and the numbers of seeds and embryos in each stage were recorded for each collection date. Surprisingly, a broad range of seed and embryo developmental stages could be found in samaras collected from the same tree on the same date, in particular for the later collection dates. Using this information, single-date collections of seeds with embryos at various stages of development were made from three local Athens, GA green ash trees and one horticultural cultivar and cultured on two different basal media with different combinations of plant growth regulators (PGRs). A low percentage of zygotic embryo explants at an intermediate stage of development from all three local source trees produced proembryogenic masses (PEMs) when cultured on a modified Woody Plant Medium with 2,4-dichlorophenoxyacetic acid and benzyladenine. Although embryogenesis was also induced from explants of the horticultural cultivar, these cultures failed to produce germinable somatic embryos. Transfer of PEMs to PGR-free medium resulted in highly dense production of somatic embryos, some of which were germinated to produce somatic seedlings.     

Application of fluorimetric analysis of plant esterases to study of programmed cell death and effects of cadmium(II) ions

Esterases (EC 3.1.1.x) represent a diverse group of hydrolases catalyzing the cleavage and formation of carboxyl ester bonds. Their connection with development has made them a suitable marker of development in plants. In the present work, we focused on the fluorimetric determination of the plant esterases in plant cell cultures (tobacco BY-2 cells and early somatic embryos of Norway spruce, clone 2/32) with respect to application the method for the study of programmed cell death and the influence of cadmium(II) ions on the plant cells. The programmed cell death has been triggered by sodium nitroprusside and glucose oxidase. The determination of the esterase activity by the proposed technique in a cell extract determined very small difference in enzyme activity, which was a reliable marker of metabolic changes. In addition, the esterase activity of spruce somatic embryos decreased with the increase in medium Cd concentration.     

The transition of proembryogenic masses to somatic embryos in Araucaria angustifolia (Bertol.) Kuntze is related to the endogenous contents of IAA,ABA and polyamines

In somatic embryogenesis (SE) of conifers, the inability of many embryogenic cell lines to form well-developed somatic embryos may results from failure and constraints during the transition of proembryogenic masses (PEMs) to early somatic embryos. In the present work, we propose the inclusion of a preculture and prematuration steps looking at enhancing PEM III-to-early somatic embryos transition. It was further hypothesized that these results would correlate with the contents of endogenous indole-3-acetic acid (IAA), abscisic acid (ABA) and polyamines (PA). To test these hypotheses, the embryogenic culture was subjected to preculture with fluridone (FLD) and prematuration treatments with different combinations of carbon source and polyethylene glycol (PEG). The frequency of PEM III was increased after FLD preculture and the contents of IAA and ABA decreased, while the contents of PA increased. Putrescine (Put) was the most abundant PA present at this stage, followed by spermidine (Spd) and spermine (Spm). In early embryogenesis, prematuration treatments supplemented with maltose or lactose plus PEG enhanced the PEM III-to-early somatic embryos transition. IAA and ABA contents increased at this stage, while a decrease of the total free PA levels was observed. Put was the most abundant PA, followed by Spd and Spm, mainly in the treatment supplemented with PEG. This resulted in a decrease of PA ratio (Put/Spd + Spm) and, hence, PEM III-to-early somatic embryos transition. It was concluded that the preculture with FLD and prematuration treatments promote the PEM III-to-early somatic embryos transition throughout the whole early developmental process in Araucaria angustifolia.     

Effects from shear stress on morphology and growth of early stages of Norway spruce somatic embryos

The shear stress effect on directional expansion of pro embryogenic masses (PEMs) and suspensor cell development of somatic embryos of Norway spruce (Picea abies) at the proliferation stage was studied by a direct and quantitative image analysis system. The experimental system allowed for detailed observations of the effect of hydrodynamic shear stress in rotating and deforming liquid cultures of proliferating Norway spruce somatic embryos. Briefly, somatic embryos at an early development stage comprised only of clusters of meristematic cells without suspensor cells were fixed on an alginate film. The alginate film was affixed on the bottom of a flow cell and the somatic embryos were subjected to laminar flow through the chamber of the flow cell. Magnified images of the cell clusters were collected every 24 h. The image data was processed based on a normalized cross‐correlation method, capable of measuring morphological and size features of individual cell clusters in both temporal and spatial domains. No suspensor cells developed in the cell clusters under shear stress of 140 s^?1 for the duration of the experiments. Cell clusters in the control cultured in stationary liquid conditions developed suspensor cells after 5–9 days in culture. Furthermore, the radial growth of meristematic cell clusters was inhibited by shear rates of 86 and 140 s^?1, corresponding to shear stress of 0.086 and 0.14 N/m², compared to growth under stationary conditions. The shear rate showed a significant negative correlation to growth rate. Control group showed no preference for direction during growth under static conditions. Biotechnol. Bioeng. 2010; 105: 588–599. © 2009 Wiley Periodicals, Inc.     

Triglycerides in embryogenic conifer calli: a comparison with zygotic embryos

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.     

Rhizobial Nod factors stimulate somatic embryo development in Picea abies

 Nod factors are lipochitooligosaccharides (LCOs) secreted by rhizobia. Nod factors trigger the nodulation programme in a compatible host. A bioassay was set up to test how crude (NGR234) and purified (NodS) Nod factors influence cell division and somatic embryogenesis in a conifer, Norway spruce (Picea abies). The Nod factors promoted cell division in the absence of auxin and cytokinin. More detailed studies showed that NodS stimulates development of proembryogenic masses from small cell aggregates and further embryo development. However, stimulation was only observed in low-density cell cultures. Our data suggest that rhizobial Nod factors substitute for conditioning factors in embryogenic cultures of Norway spruce. Received: 20 January 1999 / Revision received: 26 March 1999 / Accepted: 27 April 1999