BACKGROUND
plant somatic embryogenesis  has become an established biotechnology within the horticulture, agriculture and forest industries, providing the capability for commercial-scale production of clonal seedlings  <cit> .
however, the efficiency of inducing embryogenic tissue formation  continues to be problematic, particularly in woody species such as conifers.
for example, although zygotic embryos from a few species belonging to the pinaceae family are highly responsive, many other conifer species are either completely nonresponsive or produce efficiencies too low to be commercially viable.
an even more prominent issue is the recalcitrance of tissues from adult trees, which, if overcome, would allow unlimited propagation of individual trees with elite characteristics  <cit> .
while judicious manipulation of induction media has found success in improving se induction efficiency from zygotic embryos, particularly for pines  <cit> , successful application to vegetative tissues has to date been marginal, at best  <cit> .
in addition, although many physiological and genetic factors impacting se induction have been documented for angiosperms  <cit> , lack of an effective experimental system has impeded efforts to identify even the most fundamental aspects underpinning se induction within vegetative tissues of conifers.
in an attempt to address this deficiency, experiments initiated over a decade ago targeted somatic embryo-derived white spruce trees with the expectation that they would have a greater propensity for se induction than trees grown from seed.
this led to the identification of a clonal line of white spruce that produced shoot buds that have remained responsive to se induction even after reaching sexual maturity  <cit> .
combined with advances in conifer genomics  <cit> , this presented an unprecedented opportunity to explore the molecular aspects of se induction within shoot primordia of adult spruce trees.
using a recently constructed conifer 32 k oligo-probe microarray  <cit> , transcriptome-wide expression profiling led to the identification of four of the most differentially expressed genes within this and a nonresponsive genotype at day  <dig> of induction.
expanding the analysis to day  <dig> using absolute qpcr revealed substantive differences in the expression dynamics of these candidate genes.
most evident was that both the magnitude and duration of candidate gene expression were greater within the nonresponsive genotype, which is indicative of an intense physiological response to the induction treatment that may be antagonistic to se induction.
examination of their putative identities further revealed that this intense response may be a result of biotic defense elicitation, whereas the moderate response of the responsive genotype is suggestive of an adaptive response.
RESULTS
induction of somatic embryogenesis within primordial shoots
a detailed description of se induction within primordial shoot explants of the responsive genotype  has previously been described  <cit> .
briefly, buds were disinfected, primordial shoots excised and cut into sections before being placed onto se induction medium .
with the expectation that differential gene expression could be associated with the responsiveness  to se induction, microarray analysis was conducted with rna extracted from explants following one week of induction .
selection of this time point was based on empirical observations suggesting that it was sufficiently early to avoid biases produced by embryonal mass formation, which could confound identification of genes associated with se induction, rather than those that become active during embryogenesis.
during the first two weeks of induction, explants of both g <dig> and that of a nonresponsive genotype  were characterized by elongation of the needle primordia and formation of small amounts of callus on the cut surfaces and at the bases of elongated needle primordia.
during the third week of induction, some of the g <dig> explants produced nodules on the elongated needle primordia or within the callus, along with minute amounts of embryonal masses  that marked the initiation of se .
during the fourth week of induction, some of the g <dig> explants generated rapidly proliferating em .
after 16 weeks,  <dig> of  <dig>  g <dig> shoot explants produced em, while none of the 480 g <dig> explants responded.
microarray analysis and selection of candidate genes
the microarray analysis was conducted using explants taken at the point of collection  and after one week of se induction , with five biological replicates analyzed for each genotype per time point .
intra-genotype differences between day  <dig> and  <dig> were substantive, with  <dig> and  <dig> targets being differentially expressed within g <dig> and g <dig>  respectively .
although this includes  <dig> targets that were shared, the total number of differentially expressed targets  represents a sizeable proportion of the  <dig>  distinct white spruce genes represented on the microarray  <cit> .
an inter-genotype comparison reveals many small differences, with  <dig> targets differing significantly at day  <dig>  as compared to  <dig> targets at day  <dig> .
comparing the magnitude of intra-genotype fold-differences further supported the similarity of response to the induction treatment .
such moderate differences suggest that the induction treatment generated a largely shared response with regards to the genes involved.
however, this does not take into account quantitative differences in expression levels, an aspect that was examined during the qpcr analysis .
to further investigate how genotype and the induction treatment interacted, a two-way anova analysis was performed .
this revealed that  <dig> targets were differentially expressed across all combinations, with about 90% responding solely in relation to the se induction treatment.
furthermore, about 10% differed between the two genotypes, and about 3% showed a significant interaction between genotype and treatment.
it is important to note that all targets showing a genotype effect also showed response to the se induction, in which 37% showed a genotype x treatment interaction.
to identify candidate genes for qpcr analysis, the microarray data were sorted based on the largest fold differences relative to the other genotype at day  <dig> of induction , which showed similar trends in both the number of targets and the magnitude of differential gene expression .
with the objective of selecting four candidate genes that most greatly differentiated each genotype at day  <dig>  the most differentially expressed targets were examined in detail.
this revealed that nine of the top  <dig> within g <dig> were found to be genes belonging to a small gene family encoding for three variants of an usual conifer-specific dehydrin called dhn <dig> , which has been identified previously in norway spruce  <cit> .
due to their high degree of similarity, these dhn <dig> genes were considered to represent a single target .
of the three remaining g <dig> candidates, a putative identity was found for only one, showing a high degree of similarity to the apoplastic class iii peroxidase, atprx <dig>  from arabidopsis .
the last two candidates both encode for unusual proteins that appear to be conifer-specific, containing repetitive segments rich in threonine-glutamine and proline, respectively .
fold differences between day  <dig> and  <dig> within each genotype are also listed for each candidate gene, along with their unigene accession number.
the dhn <dig> dehydrin candidate was found to be encoding for three closely related variants .
putative identities were found for all four of the g <dig> candidates .
the top two were found to encode for closely related proteins with high levels of sequence similarity to an unusual class of serine protease inhibitor that is highly conserved throughout the angiospermae, and predicted to have an amino terminal signal peptide based on signalp  <dig>  analysis  <cit>  .
another striking feature of these protease inhibitors is the presence of eight conserved cysteine residues that conform to the crp <dig> class of small cysteine-rich peptides, a very large family of excreted peptides that include defensins, along with many other antimicrobial proteins  <cit> .
like that of the g <dig> pgprx <dig>  the third g <dig> candidate encodes for an apoplastic class iii peroxidase, but which is most similar to arabidopsis atprx <dig> .
the remaining g <dig> candidate encodes for a cell wall invertase most similar to the arabidopsis atcwinv <dig> .
fold differences between day  <dig> and  <dig> within each genotype are also listed for each candidate gene, along with their unigene accession number.
with respect to changes in expression over time, comparing day  <dig> with day  <dig> revealed that all but one of the candidate genes increased significantly within both genotypes, with no example of a reduction in gene expression in the apposing genotype.
this indicates that differential expression at day  <dig> was due to higher levels of activation within the originating genotype .
also notable is that the expression of all but one of the g <dig> candidates increased to greater levels within the g <dig> explants than that of the g <dig> candidates within the g <dig> explants, suggesting that a major distinguishing characteristic of the nonresponsive g <dig> genotype is higher levels of candidate gene activation.
concordance of microarray analysis with absolute qpcr
conducting absolute quantification greatly increased the resolution of the analysis, in addition to allowing the expression of any gene to be directly compared with that of any other gene, within and between multiple samples.
this was accomplished using a method developed by our group called lre qpcr that greatly simplifies absolute quantification, in large part by abrogating the need to construct target-specific standard curves  <cit> .
as is described in the methods section, expression analysis of nine reference genes revealed that within the five biological replicates taken for each time point used in the microarray analysis, the average variance was found to be about ±20% , which is in part indicative of the analytical precision that can be achieved with lre qpcr  <cit> .
furthermore, when their average expression level was compared across the four sample groups, six of nine references generated inter-sample group variances below ±20%, reflective of a remarkably low level of biological variability within this experimental system.
this in turn circumvented the need to conduct reference gene normalization as is commonly practiced for qpcr-based gene expression analysis, particularly for those employing relative quantification  <cit> .
table  <dig> summarizes the expression levels of the eight candidate genes, revealing that expression at day  <dig> was low for all the candidate genes within both genotypes, ranging from  <dig>  to  <dig> transcripts per 10 ng rna.
although the biological perspective of such a small dataset is necessarily limited, this does suggest that all of the candidate genes were relatively quiescent at the point of bud collection.
in addition, consistent with that predicted by the microarray analysis, activation of the g <dig> candidates within the g <dig> explants was on average 9-fold higher than that of the g <dig> candidates within the g <dig> explants.
quantities are expressed as the number of transcripts per 10 ng rna ± cv  x 100%).
individual quantifications are provided in additional file  <dig>
table  <dig> compares the relative differences in candidate gene expression between the two genotypes at day  <dig>  which provides broad confirmation of the microarray analysis.
for example, the relative ranking based on the magnitude of fold differences, as predicted by microarray and qpcr quantification, is in general agreement within and between the two groups of candidate genes.
one obvious exception is the qpcr-derived ratio for the qt-repeat candidate of 340-fold.
however, this is a result of the very low expression levels within the g <dig> explants at day  <dig> , bringing into doubt the comparability with the microarray analysis.
these datasets also reflect the limited biological perspective that can be achieved with analysis of only two time points.
a major aspect of this study was thus to exploit the high capacity of lre qpcr to expand the analysis to day  <dig> of induction.
profiling the dynamics of candidate gene expression
expanding the analysis to day  <dig> by including three additional time points  allowed the dynamics of candidate gene expression to be defined in greater detail.
for example, within the g <dig> explants the qt-repeat candidate expression reached near maximal levels by day  <dig>  a level that was maintained up to day  <dig>  whereas within the g <dig> explants, its expression was nearly absent throughout the entire induction treatment .
extensive differential expression was also revealed for the apoplastic peroxidase pgprx <dig> within the g <dig> explants, reaching maximal expression by day  <dig>  but falling 3-fold by day  <dig>  to a level that was maintained up to day  <dig> .
the dynamics of dhn <dig> expression was similar in nature to pgprx <dig>  peaking at day  <dig> followed by a progressive 3-fold reduction by day  <dig> within the g <dig> explants, indicative of an early, transient-like activation .
however, dhn <dig> expression was not only apparent within the g <dig> explants, but progressively increased up to day  <dig>  suggesting that activation of this g <dig> candidate gene is much less genotype-specific.
while differential expression of the proline-rich candidate was maintained up to day  <dig>  both genotypes generated similar expression dynamics, again reflective of modest, if any, genotypic specificity .
for g <dig>  all four candidates demonstrated high levels of differential expression .
in addition, expression for all but pgcwinv <dig> progressively increased during the induction treatment, all reaching maximal levels that were on average about 20x greater than the maximum expression of the g <dig> candidates within the g <dig> explants .
overall, the expression dynamics within the g <dig> explants is consistent with an intense and sustained response to the induction treatment.
in summary, qpcr expression profiling confirmed the efficacy of candidate gene selection based on microarray analysis at day  <dig> of induction, in that all of the candidate genes maintained differential expression within their originating genotype throughout the entire induction treatment, although to varying degrees.
in addition, the overall biological variability was sufficiently low to reveal strong trends in gene expression dynamics, and to illustrate the utility of absolute quantification, which, among other attributes, provides the ability to assess the magnitude of expression of individual genes.
also consistent with that predicted by the microarray analysis was the high level of induction of all four g <dig> candidates within the g <dig> explants, which was maintained well beyond day  <dig>  this suggests that a major distinguishing characteristic of these nonresponsive explants is an intense physiological response to the se induction treatment.
discussion
recalcitrance of plant explants to many types of tissue culture manipulation, including se induction, has long been an impediment to clonal propagation of individual plants with elite characteristics, a capability that has significant commercial implications.
these also include rare traits, such as insect or pathogen resistance, for which rapid propagation and dissemination could have important ecological implications.
this is particularly relevant to forest trees, whose long generation times pose substantive challenges to traditional propagation approaches such as rooting of cuttings, and to genetic improvement programs based on conventional breeding.
these limitations are further exacerbated by the fact that many elite traits become most evident in mature plants, a time at which many woody species, including conifers, become recalcitrant to tissue culture manipulation  <cit> .
the identification of a clonal line of adult white spruce trees that produce shoot primordia responsive to se induction thus presented a unique opportunity to address the molecular aspects of se induction, with the expectation that this may be broadly applicable to other plant species.
stress-response as a likely determinant for se induction responsiveness
as is often the case for se-induction research, a major presumption entering into this study was that genes selectively expressed within the responsive g <dig> bud explants would become the primary targets for investigation.
indeed, expression profiling revealed an unusual glutamine-threonine repeat protein  that was specifically expressed throughout the entire se induction treatment .
although such a dramatic differential expression implies a potentially important role in induction responsiveness, this protein appears to be conifer-specific with no known function, excluding the likelihood that this protein plays a universal role in somatic embryogenesis.
this is also the case for a proline-rich protein , which showed marginal, albeit persistent, differential expression in the g <dig> bud explants up to day  <dig> ; however, this protein also appears to be conifer-specific with no known function.
although the two remaining candidates encode for proteins with putative functions , the greatest differential expression occurred during early stages of the induction treatment , long before embryogenic tissues begin to emerge, which again does not greatly support a role, at least directly, in determining se induction responsiveness.
in contrast to this temperate response, the intense candidate gene activation within the nonresponsive g <dig> bud explants was not only found to persist into the late stages of the induction treatment, but also reached very high levels  .
while the limited use of absolute quantification has to date provided an inadequate context for universally comparing gene expression levels, ef1α does provide some perspective as to the magnitude of candidate gene expression.
utilized as a reference gene, ef1α expression averaged around  <dig>  transcripts per 10 ng rna , which is the most highly expressed gene encountered in this study.
this is consistent with it being one of the most highly expressed genes in developing conifer shoots, as based on est clone frequency and microarray analysis .
it is thus the magnitude of candidate gene expression within the nonresponsive g <dig> explants that suggests the physiological response of a primordial shoot explant, particularly during the early stages of se induction, could determine its ability to become responsive to se induction.
while the subject area of stress physiology is vast, it is apparent that a number of general principles described for angiosperms could provide insights into the physiological response of conifer bud explants, particularly in relation to abiotic and biotic defense responses.
for example, a transient oxidative burst of low intensity is predicted to occur immediately after an explant is introduced into culture, which is indicative of early oxidative signaling generated during both abiotic stress  and biotic defense responses  <cit> .
a major distinction, however, is that elicitation of a biotic defense response is associated with a second prolonged oxidative burst of high intensity, which in turn triggers massive transcriptional and metabolic reprogramming, including high level induction of defense protein expression, slowing of growth, and, in the most extreme cases, induced cell death  <cit> .
this possess the question as to whether the intense response generated by the g <dig> explants is reflective of a biotic defense response that makes them physiologically recalcitrant to se induction, in contrast to an adaptive stress response within the g <dig> explants that generated a physiological state conducive to se induction.
although such generalizations provide an attractive model, it should be stressed that angiosperm defense responses have been found to be highly dynamic and complex processes that involve cross-talk between signaling networks regulated by salicylate, jasmonates, and ethylene, in combination with other plant hormones  <cit> .
it is therefore difficult to draw specific parallels to conifer bud explants without direct supporting evidence.
nevertheless, examining the putative functions of the proteins encoded by the four g <dig> candidate genes  provides support for the contention that the se induction treatment elicited a biotic defense response within these nonresponsive explants.
apoplastic class iii peroxidases play a prominent role in biotic defense activation
class iii peroxidases have been found to generate apoplastic hydrogen peroxide that acts as a signal for biotic defense elicitation  <cit> .
direct demonstration of this was recently reported for maize, in which u. maydis  leaf infection was found to induce the expression of a single class iii peroxidase gene , and that virulence of this pathogen is dependent on its excretion of a peroxidase inhibitor peptide   <cit> .
furthermore, induced gene silencing of pox  <dig> was found to restore virulence even in the absence of pep  <dig>  providing direct evidence that pox <dig> activity is essential for mounting a successful biotic defense response against this pathogen  <cit> .
the progressive, high level activation of a class iii peroxidase within the g <dig> explants  is thus consistent with a role in biotic defense elicitation.
additional, albeit tentative, support for such a contention is that this g <dig> candidate is most similar in amino acid sequence to the arabidopsis class iii peroxidase, atprx <dig> , which belongs to an unusual evolutionary branch of plant peroxidases  <cit> .
increased expression of atprx <dig> produced by wounding and microbial attack has led to the suggestion that it has a protective role against pathogens  <cit> .
a more direct demonstration of a role in biotic defense comes from the fact that overexpression of atprx <dig> in arabidopsis produces resistance to botrytis cinerea <cit> .
thus, currently available data suggests that persistent, high level expression of an apoplastic class iii peroxidase gene plays a central role in biotic defense activation, a role that the g <dig> pgprx <dig> may play in conifers.
conversely, the transient nature of the g <dig> class iii peroxidase pgprx <dig> gene activation  could be reflective of an adaptive stress response in which the initial oxidative burst dissipates during the first few days of induction treatment, restoring cellular redox homeostasis  <cit> .
this is similar to the activation, although slightly later, of the g <dig> dehydrin dhn <dig> gene , which is a conifer-specific dehydrin that has been reported to play a role in bud dormancy and overwintering in norway spruce  <cit> .
this too supports the contention that the g <dig> explants elicited an adaptive stress response, in that dehydrins have long been recognized as playing a fundamental role in adapting to environmental stresses  <cit> .
thus, although speculative, it could be argued that an adaptive stress response may be an important determinant for establishing se induction responsiveness.
induction of a cell wall invertase
activation of cell wall invertases, which catalyze the hydrolysis of sucrose to glucose and fructose within the apoplast, have also been found to play a prominent role in biotic defense by providing the large quantities of energy required for mounting an intense metabolic response  <cit> .
rnai inhibition of expression has provided direct support for a central role of a cell wall invertase in defense response elicitation in tobacco  <cit> .
indeed, the prominence of cell wall invertase induction has led to a proposal that it be classified as a pathogenesis-related protein  <cit> , which is a large class of defense proteins that become highly expressed following biotic defense activation  <cit> .
amino acid sequence comparison to arabidopsis revealed that the g <dig> invertase is most similar to atcwinv <dig>  , which is a member of a small gene family within arabidopsis  <cit> .
a number of studies have reported induction of atcwinv <dig> expression following pathogen infection of arabidopsis leaves  <cit> , in addition to induction following agrobacterium infection of arabidopsis cell cultures  <cit> .
importantly, selective induction of atcwinv <dig> following wounding also suggests a general role in adaptive stress responses  <cit> .
this is consistent with the progressive increase in expression of pgcwinv <dig> observed within the g <dig> explants.
however, this is contrasted by a rapid activation within the g <dig> explants between day  <dig> and  <dig>  reaching an average of about twice that found within the g <dig> explants, a level that is maintained up to day  <dig> of the induction treatment .
this could be reflective of a more intense metabolic response of the g <dig> explants during the earliest stages of the induction treatment, a presumption consistent with the high levels of expression observed for the two remaining g <dig> candidate genes.
activation of two i <dig> serine protease inhibitors
the two most highly expressed g <dig> candidate genes were found to encode for closely related protease inhibitors belonging to an unassigned subclass of the merops i <dig> family of serine protease inhibitors  <cit> .
this subclass has a number of distinguishing features, including the presence of a transient peptide, an unusually small mature protein , and the presence of eight highly conserved cysteines .
in fact, these features have led to their classification into the superfamily of small cysteine-rich peptides , a very large family of secreted peptides composed of several hundred genes within arabidopsis.
initially founded on structural similarities with defensins, which is an ancient form of antimicrobial peptide  <cit> , a large number of crps have been shown to play a role in biotic defense  <cit> .
another distinctive characteristic of this subclass of i <dig> protease inhibitors is the occurrence of closely related homologues throughout the angiospermae, principally as a single gene  <cit> .
in fact, this high level of conservation led hartl et al.
<cit>  to suggest that in addition to a probable role in biotic defense, they could have an essential role in plant physiology.
the presence of two highly conserved homologs within conifers provides support for such a possibility.
a direct link of the angiosperm homolog to biotic defense activation comes from expression analysis of u. maydis Δpep1-infected maize leaves, which elicits a massive biotic defense response, as was described earlier.
among the  <dig> genes found to be induced, the maize i <dig> homolog  was the fourth most highly induced, increasing by 166-fold within  <dig> h post-inoculation .
similarly, the arabidopsis i <dig> homolog  was the fifth most induced gene following infection by trichoderma harzianum, reaching a  <dig> -fold increase within  <dig> h  <cit> .
application of biotic defense elicitors to 10-day-old arabidopsis seedlings was also found to induce this i <dig> gene, with the bacterial flagellin peptide flg <dig> generating  <dig> - and  <dig> -fold increases after  <dig> and  <dig> h, respectively .
oxidative stress induction of the maize homolog was also demonstrated in maize leaves , reaching  <dig> - and  <dig> -fold increases  <dig> h after application of h2o <dig> and methyl viologen, respectively  <cit> .
however, a role in abiotic defense is unlikely, as ectopic expression of the arabidopsis homolog in transgenic seedlings dramatically reduced resistance to oxidative, osmotic and salt stress  <cit> .
the induction kinetics of the two conifer i <dig> homologs within the g <dig> explants is thus consistent with biotic defense activation as observed in angiosperms, with both genes reaching close to  <dig>  transcripts per 10 ng rna by day  <dig>  which, in comparison with day  <dig>  roughly represents a 2000-fold increase.
another notable feature was a progressive increase in expression throughout the entire induction treatment, reaching about  <dig>  transcripts per 10 ng rna by day  <dig> .
thus, while the precise biochemical function of this unusual class of serine protease inhibitors remains to be determined, the high level of amino acid sequence conservation, combined with their expression dynamics, provide support to the supposition that these two conifer protease inhibitors play a role in conifer biotic defense, similar to that observed in angiosperms.
a paradigm shift towards physiological processes that may antagonize se induction
while it has long been recognized that the physiological state of an explant can be a major determinant for responsiveness to se induction, very little is understood about the underlying mechanisms.
furthermore, the vast majority of research efforts have historically focused on defining factors that promote se induction, for example, through the activation of se-promoting genes.
this study expands this paradigm, suggesting that nonresponsiveness of an explant is not necessarily due to an innate lack of se promoting activity, but that biotic defense activation could potentially be a dominant antagonist.
a number of physiological aspects of biotic defense elicitation provide general support for such a presumption.
for example, activation of biotic defense through exogenous application of various elicitors has been shown to dramatically reduce plant growth, a result of redirecting metabolic energy from growth to defense  <cit> .
this includes the action of jasmonates , the central regulators of biotic defense elicitation associated with wounding, which have been shown to directly mediate a switch from growth to the production of biocidal compounds, cell-wall remodeling, and defense protein expression  <cit> .
indeed, growth repression by jas has been directly linked to inhibition of cell-cycle progression  <cit> , in addition to directly antagonizing the growth promoting activity of gibberellic acid  <cit> , thus providing evidence that jas could be direct antagonists of se induction, based in part on the assumption that cell division is necessary for embryogenic tissue formation.
another line of supporting evidence, albeit indirect, comes from proteomic studies that have reported a correlation between expression of biotic defense proteins, primarily pathogenesis-related proteins, and a lack of embryogenic competency of tissues in culture  <cit> .
another notable observation related to the quantitative nature of proteomic analysis, is the magnitude of defense protein expression within these nonembryogenic tissues, often being the most prominent proteins in the analysis.
while it is difficult to draw a direct comparison, this is consistent with the intense activation of the g <dig> candidate genes within the g <dig> explants, and with the supposition that redirecting metabolic resources towards the production of such large quantities of defense proteins could itself be antagonistic to the formation of embryogenic tissues.
se induction within leaf explants from the model legume medicago truncatula, an experimental system similar to the bud explants used in this study, has also been used to directly compare responsive and nonresponsive genotypes  <cit> .
proteomic analysis revealed large physiological differences as reflected by high levels of protein accumulation, some of which were identified as stress proteins, within a nonresponsive line during the first week of the se induction treatment.
although an association with biotic defense elicitation was not evident from the data presented, this led the authors to suggest that a hyperresponse to the stress produced by the induction treatment could be related to a lack of responsiveness  <cit> .
this is a scenario similar to the intense physiological response of the g <dig> explants, and reminiscent of the high levels of defense protein accumulation within nonembryogenic tissues, as discussed above.
finally, although this study provides evidence for a link between biotic defense elicitation and recalcitrance to se induction, it has also generated hypotheses that could not be directly addressed within the experimental design.
first, only one nonresponsive genotype was analyzed, so the question as to whether a similar physiological response occurs in other nonresponsive genotypes remains unanswered.
second, the hypothesis of a direct association between biotic defense elicitation and suppression of se induction remains to be tested.
thus, it is possible that the differences in physiological response observed within the g <dig> and g <dig> explants is a result of a genotypic difference unrelated to se responsiveness.
third, it is unknown whether se could be induced in the g <dig> explants even if biotic defense elicitation were to be mitigated.
additional work is thus required before a definitive understanding of the broad applicability and implications of these findings can be achieved.
CONCLUSIONS
the central conclusion of this study is that the physiological response of conifer bud explants, particularly in relation to elicitation of a defense response, could be an important determinant of se induction responsiveness.
although definitive demonstration that biotic defense activation is antagonistic to se induction requires additional evidence, many general characteristics, such as the dramatic metabolic and transcriptional reprogramming associated with its elicitation, support a role contributing to the recalcitrance of explants to se induction.
in addition, it opens new avenues of investigation into the mechanisms regulating the activation and intensity of defense responses within explants placed into culture, along with the prospective of developing methods that could be used to suppress them, with the expectation that this could generate a physiological state more conducive not only to se induction, but potentially to other types of tissue culture manipulation.

 plant somatic embryogenesis has become an established biotechnology within the horticulture, agriculture and forest industries, providing the capability for commercial-scale production of clonal seedlings. however, the efficiency of inducing embryogenic tissue formation continues to be problematic, particularly in woody species such as conifers. for example, although zygotic embryos from a few species belonging to the pinaceae family are highly responsive, many other conifer species are either completely nonresponsive or produce efficiencies too low to be commercially viable. an even more prominent issue is the recalcitrance of tissues from adult trees, which, if overcome, would allow unlimited propagation of individual trees with elite characteristics. while judicious manipulation of induction media has found success in improving se induction efficiency from zygotic embryos, particularly for pines, successful application to vegetative tissues has to date been marginal, at best. in addition, although many physiological and genetic factors impacting se induction have been documented for angiosperms, lack of an effective experimental system has impeded efforts to identify even the most fundamental aspects underpinning se induction within vegetative tissues of conifers. in an attempt to address this deficiency, experiments initiated over a decade ago targeted somatic embryo-derived white spruce trees with the expectation that they would have a greater propensity for se induction than trees grown from seed. this led to the identification of a clonal line of white spruce that produced shoot buds that have remained responsive to se induction even after reaching sexual maturity. combined with advances in conifer genomics, this presented an unprecedented opportunity to explore the molecular aspects of se induction within shoot primordia of adult spruce trees. using a recently constructed conifer 32 k oligo-probe microarray, transcriptome-wide expression profiling led to the identification of four of the most differentially expressed genes within this and a nonresponsive genotype at day of induction. expanding the analysis to day using absolute qpcr revealed substantive differences in the expression dynamics of these candidate genes. most evident was that both the magnitude and duration of candidate gene expression were greater within the nonresponsive genotype, which is indicative of an intense physiological response to the induction treatment that may be antagonistic to se induction. examination of their putative identities further revealed that this intense response may be a result of biotic defense elicitation, whereas the moderate response of the responsive genotype is suggestive of an adaptive response.  induction of somatic embryogenesis within primordial shoots a detailed description of se induction within primordial shoot explants of the responsive genotype has previously been described. briefly, buds were disinfected, primordial shoots excised and cut into sections before being placed onto se induction medium. with the expectation that differential gene expression could be associated with the responsiveness to se induction, microarray analysis was conducted with rna extracted from explants following one week of induction. selection of this time point was based on empirical observations suggesting that it was sufficiently early to avoid biases produced by embryonal mass formation, which could confound identification of genes associated with se induction, rather than those that become active during embryogenesis. during the first two weeks of induction, explants of both g and that of a nonresponsive genotype were characterized by elongation of the needle primordia and formation of small amounts of callus on the cut surfaces and at the bases of elongated needle primordia. during the third week of induction, some of the g explants produced nodules on the elongated needle primordia or within the callus, along with minute amounts of embryonal masses that marked the initiation of se. during the fourth week of induction, some of the g explants generated rapidly proliferating em. after 16 weeks, of g shoot explants produced em, while none of the 480 g explants responded. microarray analysis and selection of candidate genes the microarray analysis was conducted using explants taken at the point of collection and after one week of se induction, with five biological replicates analyzed for each genotype per time point. intra-genotype differences between day and were substantive, with and targets being differentially expressed within g and g respectively. although this includes targets that were shared, the total number of differentially expressed targets represents a sizeable proportion of the distinct white spruce genes represented on the microarray. an inter-genotype comparison reveals many small differences, with targets differing significantly at day as compared to targets at day. comparing the magnitude of intra-genotype fold-differences further supported the similarity of response to the induction treatment. such moderate differences suggest that the induction treatment generated a largely shared response with regards to the genes involved. however, this does not take into account quantitative differences in expression levels, an aspect that was examined during the qpcr analysis. to further investigate how genotype and the induction treatment interacted, a two-way anova analysis was performed. this revealed that targets were differentially expressed across all combinations, with about 90% responding solely in relation to the se induction treatment. furthermore, about 10% differed between the two genotypes, and about 3% showed a significant interaction between genotype and treatment. it is important to note that all targets showing a genotype effect also showed response to the se induction, in which 37% showed a genotype x treatment interaction. to identify candidate genes for qpcr analysis, the microarray data were sorted based on the largest fold differences relative to the other genotype at day of induction, which showed similar trends in both the number of targets and the magnitude of differential gene expression. with the objective of selecting four candidate genes that most greatly differentiated each genotype at day the most differentially expressed targets were examined in detail. this revealed that nine of the top within g were found to be genes belonging to a small gene family encoding for three variants of an usual conifer-specific dehydrin called dhn, which has been identified previously in norway spruce. due to their high degree of similarity, these dhn genes were considered to represent a single target. of the three remaining g candidates, a putative identity was found for only one, showing a high degree of similarity to the apoplastic class iii peroxidase, atprx from arabidopsis. the last two candidates both encode for unusual proteins that appear to be conifer-specific, containing repetitive segments rich in threonine-glutamine and proline, respectively. fold differences between day and within each genotype are also listed for each candidate gene, along with their unigene accession number. the dhn dehydrin candidate was found to be encoding for three closely related variants. putative identities were found for all four of the g candidates. the top two were found to encode for closely related proteins with high levels of sequence similarity to an unusual class of serine protease inhibitor that is highly conserved throughout the angiospermae, and predicted to have an amino terminal signal peptide based on signalp analysis. another striking feature of these protease inhibitors is the presence of eight conserved cysteine residues that conform to the crp class of small cysteine-rich peptides, a very large family of excreted peptides that include defensins, along with many other antimicrobial proteins. like that of the g pgprx the third g candidate encodes for an apoplastic class iii peroxidase, but which is most similar to arabidopsis atprx. the remaining g candidate encodes for a cell wall invertase most similar to the arabidopsis atcwinv. fold differences between day and within each genotype are also listed for each candidate gene, along with their unigene accession number. with respect to changes in expression over time, comparing day with day revealed that all but one of the candidate genes increased significantly within both genotypes, with no example of a reduction in gene expression in the apposing genotype. this indicates that differential expression at day was due to higher levels of activation within the originating genotype. also notable is that the expression of all but one of the g candidates increased to greater levels within the g explants than that of the g candidates within the g explants, suggesting that a major distinguishing characteristic of the nonresponsive g genotype is higher levels of candidate gene activation. concordance of microarray analysis with absolute qpcr conducting absolute quantification greatly increased the resolution of the analysis, in addition to allowing the expression of any gene to be directly compared with that of any other gene, within and between multiple samples. this was accomplished using a method developed by our group called lre qpcr that greatly simplifies absolute quantification, in large part by abrogating the need to construct target-specific standard curves. as is described in the methods section, expression analysis of nine reference genes revealed that within the five biological replicates taken for each time point used in the microarray analysis, the average variance was found to be about ±20%, which is in part indicative of the analytical precision that can be achieved with lre qpcr. furthermore, when their average expression level was compared across the four sample groups, six of nine references generated inter-sample group variances below ±20%, reflective of a remarkably low level of biological variability within this experimental system. this in turn circumvented the need to conduct reference gene normalization as is commonly practiced for qpcr-based gene expression analysis, particularly for those employing relative quantification. table  summarizes the expression levels of the eight candidate genes, revealing that expression at day was low for all the candidate genes within both genotypes, ranging from to transcripts per 10 ng rna. although the biological perspective of such a small dataset is necessarily limited, this does suggest that all of the candidate genes were relatively quiescent at the point of bud collection. in addition, consistent with that predicted by the microarray analysis, activation of the g candidates within the g explants was on average 9-fold higher than that of the g candidates within the g explants. quantities are expressed as the number of transcripts per 10 ng rna ± cv x 100%). individual quantifications are provided in additional file table  compares the relative differences in candidate gene expression between the two genotypes at day which provides broad confirmation of the microarray analysis. for example, the relative ranking based on the magnitude of fold differences, as predicted by microarray and qpcr quantification, is in general agreement within and between the two groups of candidate genes. one obvious exception is the qpcr-derived ratio for the qt-repeat candidate of 340-fold. however, this is a result of the very low expression levels within the g explants at day, bringing into doubt the comparability with the microarray analysis. these datasets also reflect the limited biological perspective that can be achieved with analysis of only two time points. a major aspect of this study was thus to exploit the high capacity of lre qpcr to expand the analysis to day of induction. profiling the dynamics of candidate gene expression expanding the analysis to day by including three additional time points allowed the dynamics of candidate gene expression to be defined in greater detail. for example, within the g explants the qt-repeat candidate expression reached near maximal levels by day a level that was maintained up to day whereas within the g explants, its expression was nearly absent throughout the entire induction treatment. extensive differential expression was also revealed for the apoplastic peroxidase pgprx within the g explants, reaching maximal expression by day but falling 3-fold by day to a level that was maintained up to day. the dynamics of dhn expression was similar in nature to pgprx peaking at day followed by a progressive 3-fold reduction by day within the g explants, indicative of an early, transient-like activation. however, dhn expression was not only apparent within the g explants, but progressively increased up to day suggesting that activation of this g candidate gene is much less genotype-specific. while differential expression of the proline-rich candidate was maintained up to day both genotypes generated similar expression dynamics, again reflective of modest, if any, genotypic specificity. for g all four candidates demonstrated high levels of differential expression. in addition, expression for all but pgcwinv progressively increased during the induction treatment, all reaching maximal levels that were on average about 20x greater than the maximum expression of the g candidates within the g explants. overall, the expression dynamics within the g explants is consistent with an intense and sustained response to the induction treatment. in summary, qpcr expression profiling confirmed the efficacy of candidate gene selection based on microarray analysis at day of induction, in that all of the candidate genes maintained differential expression within their originating genotype throughout the entire induction treatment, although to varying degrees. in addition, the overall biological variability was sufficiently low to reveal strong trends in gene expression dynamics, and to illustrate the utility of absolute quantification, which, among other attributes, provides the ability to assess the magnitude of expression of individual genes. also consistent with that predicted by the microarray analysis was the high level of induction of all four g candidates within the g explants, which was maintained well beyond day this suggests that a major distinguishing characteristic of these nonresponsive explants is an intense physiological response to the se induction treatment. discussion recalcitrance of plant explants to many types of tissue culture manipulation, including se induction, has long been an impediment to clonal propagation of individual plants with elite characteristics, a capability that has significant commercial implications. these also include rare traits, such as insect or pathogen resistance, for which rapid propagation and dissemination could have important ecological implications. this is particularly relevant to forest trees, whose long generation times pose substantive challenges to traditional propagation approaches such as rooting of cuttings, and to genetic improvement programs based on conventional breeding. these limitations are further exacerbated by the fact that many elite traits become most evident in mature plants, a time at which many woody species, including conifers, become recalcitrant to tissue culture manipulation. the identification of a clonal line of adult white spruce trees that produce shoot primordia responsive to se induction thus presented a unique opportunity to address the molecular aspects of se induction, with the expectation that this may be broadly applicable to other plant species. stress-response as a likely determinant for se induction responsiveness as is often the case for se-induction research, a major presumption entering into this study was that genes selectively expressed within the responsive g bud explants would become the primary targets for investigation. indeed, expression profiling revealed an unusual glutamine-threonine repeat protein that was specifically expressed throughout the entire se induction treatment. although such a dramatic differential expression implies a potentially important role in induction responsiveness, this protein appears to be conifer-specific with no known function, excluding the likelihood that this protein plays a universal role in somatic embryogenesis. this is also the case for a proline-rich protein, which showed marginal, albeit persistent, differential expression in the g bud explants up to day; however, this protein also appears to be conifer-specific with no known function. although the two remaining candidates encode for proteins with putative functions, the greatest differential expression occurred during early stages of the induction treatment, long before embryogenic tissues begin to emerge, which again does not greatly support a role, at least directly, in determining se induction responsiveness. in contrast to this temperate response, the intense candidate gene activation within the nonresponsive g bud explants was not only found to persist into the late stages of the induction treatment, but also reached very high levels. while the limited use of absolute quantification has to date provided an inadequate context for universally comparing gene expression levels, ef1α does provide some perspective as to the magnitude of candidate gene expression. utilized as a reference gene, ef1α expression averaged around transcripts per 10 ng rna, which is the most highly expressed gene encountered in this study. this is consistent with it being one of the most highly expressed genes in developing conifer shoots, as based on est clone frequency and microarray analysis. it is thus the magnitude of candidate gene expression within the nonresponsive g explants that suggests the physiological response of a primordial shoot explant, particularly during the early stages of se induction, could determine its ability to become responsive to se induction. while the subject area of stress physiology is vast, it is apparent that a number of general principles described for angiosperms could provide insights into the physiological response of conifer bud explants, particularly in relation to abiotic and biotic defense responses. for example, a transient oxidative burst of low intensity is predicted to occur immediately after an explant is introduced into culture, which is indicative of early oxidative signaling generated during both abiotic stress and biotic defense responses. a major distinction, however, is that elicitation of a biotic defense response is associated with a second prolonged oxidative burst of high intensity, which in turn triggers massive transcriptional and metabolic reprogramming, including high level induction of defense protein expression, slowing of growth, and, in the most extreme cases, induced cell death. this possess the question as to whether the intense response generated by the g explants is reflective of a biotic defense response that makes them physiologically recalcitrant to se induction, in contrast to an adaptive stress response within the g explants that generated a physiological state conducive to se induction. although such generalizations provide an attractive model, it should be stressed that angiosperm defense responses have been found to be highly dynamic and complex processes that involve cross-talk between signaling networks regulated by salicylate, jasmonates, and ethylene, in combination with other plant hormones. it is therefore difficult to draw specific parallels to conifer bud explants without direct supporting evidence. nevertheless, examining the putative functions of the proteins encoded by the four g candidate genes provides support for the contention that the se induction treatment elicited a biotic defense response within these nonresponsive explants. apoplastic class iii peroxidases play a prominent role in biotic defense activation class iii peroxidases have been found to generate apoplastic hydrogen peroxide that acts as a signal for biotic defense elicitation. direct demonstration of this was recently reported for maize, in which u. maydis leaf infection was found to induce the expression of a single class iii peroxidase gene, and that virulence of this pathogen is dependent on its excretion of a peroxidase inhibitor peptide. furthermore, induced gene silencing of pox was found to restore virulence even in the absence of pep providing direct evidence that pox activity is essential for mounting a successful biotic defense response against this pathogen. the progressive, high level activation of a class iii peroxidase within the g explants is thus consistent with a role in biotic defense elicitation. additional, albeit tentative, support for such a contention is that this g candidate is most similar in amino acid sequence to the arabidopsis class iii peroxidase, atprx, which belongs to an unusual evolutionary branch of plant peroxidases. increased expression of atprx produced by wounding and microbial attack has led to the suggestion that it has a protective role against pathogens. a more direct demonstration of a role in biotic defense comes from the fact that overexpression of atprx in arabidopsis produces resistance to botrytis cinerea. thus, currently available data suggests that persistent, high level expression of an apoplastic class iii peroxidase gene plays a central role in biotic defense activation, a role that the g pgprx may play in conifers. conversely, the transient nature of the g class iii peroxidase pgprx gene activation could be reflective of an adaptive stress response in which the initial oxidative burst dissipates during the first few days of induction treatment, restoring cellular redox homeostasis. this is similar to the activation, although slightly later, of the g dehydrin dhn gene, which is a conifer-specific dehydrin that has been reported to play a role in bud dormancy and overwintering in norway spruce. this too supports the contention that the g explants elicited an adaptive stress response, in that dehydrins have long been recognized as playing a fundamental role in adapting to environmental stresses. thus, although speculative, it could be argued that an adaptive stress response may be an important determinant for establishing se induction responsiveness. induction of a cell wall invertase activation of cell wall invertases, which catalyze the hydrolysis of sucrose to glucose and fructose within the apoplast, have also been found to play a prominent role in biotic defense by providing the large quantities of energy required for mounting an intense metabolic response. rnai inhibition of expression has provided direct support for a central role of a cell wall invertase in defense response elicitation in tobacco. indeed, the prominence of cell wall invertase induction has led to a proposal that it be classified as a pathogenesis-related protein, which is a large class of defense proteins that become highly expressed following biotic defense activation. amino acid sequence comparison to arabidopsis revealed that the g invertase is most similar to atcwinv, which is a member of a small gene family within arabidopsis. a number of studies have reported induction of atcwinv expression following pathogen infection of arabidopsis leaves, in addition to induction following agrobacterium infection of arabidopsis cell cultures. importantly, selective induction of atcwinv following wounding also suggests a general role in adaptive stress responses. this is consistent with the progressive increase in expression of pgcwinv observed within the g explants. however, this is contrasted by a rapid activation within the g explants between day and reaching an average of about twice that found within the g explants, a level that is maintained up to day of the induction treatment. this could be reflective of a more intense metabolic response of the g explants during the earliest stages of the induction treatment, a presumption consistent with the high levels of expression observed for the two remaining g candidate genes. activation of two i serine protease inhibitors the two most highly expressed g candidate genes were found to encode for closely related protease inhibitors belonging to an unassigned subclass of the merops i family of serine protease inhibitors. this subclass has a number of distinguishing features, including the presence of a transient peptide, an unusually small mature protein, and the presence of eight highly conserved cysteines. in fact, these features have led to their classification into the superfamily of small cysteine-rich peptides, a very large family of secreted peptides composed of several hundred genes within arabidopsis. initially founded on structural similarities with defensins, which is an ancient form of antimicrobial peptide, a large number of crps have been shown to play a role in biotic defense. another distinctive characteristic of this subclass of i protease inhibitors is the occurrence of closely related homologues throughout the angiospermae, principally as a single gene. in fact, this high level of conservation led hartl et al. to suggest that in addition to a probable role in biotic defense, they could have an essential role in plant physiology. the presence of two highly conserved homologs within conifers provides support for such a possibility. a direct link of the angiosperm homolog to biotic defense activation comes from expression analysis of u. maydis Δpep1-infected maize leaves, which elicits a massive biotic defense response, as was described earlier. among the genes found to be induced, the maize i homolog was the fourth most highly induced, increasing by 166-fold within h post-inoculation. similarly, the arabidopsis i homolog was the fifth most induced gene following infection by trichoderma harzianum, reaching a -fold increase within h. application of biotic defense elicitors to 10-day-old arabidopsis seedlings was also found to induce this i gene, with the bacterial flagellin peptide flg generating - and -fold increases after and h, respectively. oxidative stress induction of the maize homolog was also demonstrated in maize leaves, reaching - and -fold increases h after application of h2o and methyl viologen, respectively. however, a role in abiotic defense is unlikely, as ectopic expression of the arabidopsis homolog in transgenic seedlings dramatically reduced resistance to oxidative, osmotic and salt stress. the induction kinetics of the two conifer i homologs within the g explants is thus consistent with biotic defense activation as observed in angiosperms, with both genes reaching close to transcripts per 10 ng rna by day which, in comparison with day roughly represents a 2000-fold increase. another notable feature was a progressive increase in expression throughout the entire induction treatment, reaching about transcripts per 10 ng rna by day. thus, while the precise biochemical function of this unusual class of serine protease inhibitors remains to be determined, the high level of amino acid sequence conservation, combined with their expression dynamics, provide support to the supposition that these two conifer protease inhibitors play a role in conifer biotic defense, similar to that observed in angiosperms. a paradigm shift towards physiological processes that may antagonize se induction while it has long been recognized that the physiological state of an explant can be a major determinant for responsiveness to se induction, very little is understood about the underlying mechanisms. furthermore, the vast majority of research efforts have historically focused on defining factors that promote se induction, for example, through the activation of se-promoting genes. this study expands this paradigm, suggesting that nonresponsiveness of an explant is not necessarily due to an innate lack of se promoting activity, but that biotic defense activation could potentially be a dominant antagonist. a number of physiological aspects of biotic defense elicitation provide general support for such a presumption. for example, activation of biotic defense through exogenous application of various elicitors has been shown to dramatically reduce plant growth, a result of redirecting metabolic energy from growth to defense. this includes the action of jasmonates, the central regulators of biotic defense elicitation associated with wounding, which have been shown to directly mediate a switch from growth to the production of biocidal compounds, cell-wall remodeling, and defense protein expression. indeed, growth repression by jas has been directly linked to inhibition of cell-cycle progression, in addition to directly antagonizing the growth promoting activity of gibberellic acid, thus providing evidence that jas could be direct antagonists of se induction, based in part on the assumption that cell division is necessary for embryogenic tissue formation. another line of supporting evidence, albeit indirect, comes from proteomic studies that have reported a correlation between expression of biotic defense proteins, primarily pathogenesis-related proteins, and a lack of embryogenic competency of tissues in culture. another notable observation related to the quantitative nature of proteomic analysis, is the magnitude of defense protein expression within these nonembryogenic tissues, often being the most prominent proteins in the analysis. while it is difficult to draw a direct comparison, this is consistent with the intense activation of the g candidate genes within the g explants, and with the supposition that redirecting metabolic resources towards the production of such large quantities of defense proteins could itself be antagonistic to the formation of embryogenic tissues. se induction within leaf explants from the model legume medicago truncatula, an experimental system similar to the bud explants used in this study, has also been used to directly compare responsive and nonresponsive genotypes. proteomic analysis revealed large physiological differences as reflected by high levels of protein accumulation, some of which were identified as stress proteins, within a nonresponsive line during the first week of the se induction treatment. although an association with biotic defense elicitation was not evident from the data presented, this led the authors to suggest that a hyperresponse to the stress produced by the induction treatment could be related to a lack of responsiveness. this is a scenario similar to the intense physiological response of the g explants, and reminiscent of the high levels of defense protein accumulation within nonembryogenic tissues, as discussed above. finally, although this study provides evidence for a link between biotic defense elicitation and recalcitrance to se induction, it has also generated hypotheses that could not be directly addressed within the experimental design. first, only one nonresponsive genotype was analyzed, so the question as to whether a similar physiological response occurs in other nonresponsive genotypes remains unanswered. second, the hypothesis of a direct association between biotic defense elicitation and suppression of se induction remains to be tested. thus, it is possible that the differences in physiological response observed within the g and g explants is a result of a genotypic difference unrelated to se responsiveness. third, it is unknown whether se could be induced in the g explants even if biotic defense elicitation were to be mitigated. additional work is thus required before a definitive understanding of the broad applicability and implications of these findings can be achieved.  the central conclusion of this study is that the physiological response of conifer bud explants, particularly in relation to elicitation of a defense response, could be an important determinant of se induction responsiveness. although definitive demonstration that biotic defense activation is antagonistic to se induction requires additional evidence, many general characteristics, such as the dramatic metabolic and transcriptional reprogramming associated with its elicitation, support a role contributing to the recalcitrance of explants to se induction. in addition, it opens new avenues of investigation into the mechanisms regulating the activation and intensity of defense responses within explants placed into culture, along with the prospective of developing methods that could be used to suppress them, with the expectation that this could generate a physiological state more conducive not only to se induction, but potentially to other types of tissue culture manipulation. 
most striking was that both the magnitude and duration of candidate gene expression within the nonresponsive genotype was indicative of an intense physiological response. in contrast, the more temperate response within the responsive genotype, including induction of a conifer-specific dehydrin, is more consistent with elicitation of an adaptive stress response. while additional evidence is required to definitively establish an association between se responsiveness and a specific physiological response, these results suggest that biotic defense activation may be antagonistic, likely related to the massive transcriptional and metabolic reprogramming that it elicits. microarray analysis was used to conduct transcriptome-wide expression profiling of shoot explants taken from this responsive genotype following one week of se induction, which when compared with that of a nonresponsive genotype, led to the identification of four of the most differentially expressed genes within each genotype. conifergene expression profilingmicroarray analysisabsolute qpcrlre qpcrclonal propagation identification of a clonal line of somatic embryo-derived trees whose shoot primordia have remained responsive to se induction for over a decade, provided a unique opportunity to examine the molecular aspects underpinning se within shoot tissues of adult white spruce trees. examining their putative identities further revealed that all four encoded for proteins with similarity to angiosperm proteins known to play prominent roles in biotic defense, and that their high-level induction over an extended period is consistent with activation of a biotic defense response. using absolute qpcr to expand the analysis to three weeks of induction revealed that differential expression of all eight candidate genes was maintained to the end of the induction treatment, albeit to differing degrees. a major issue for future work will be to determine how and if suppressing biotic defense activation could be used to promote a physiological state more conducive to se induction. among the many commercial opportunities afforded by somatic embryogenesis, it is the ability to clonally propagate individual plants with rare or elite traits that has some of the most significant implications. this is particularly true for many long-lived species, such as conifers, but whose long generation times pose substantive challenges, including increased recalcitrance for se as plants age.
 among the many commercial opportunities afforded by somatic embryogenesis, it is the ability to clonally propagate individual plants with rare or elite traits that has some of the most significant implications. this is particularly true for many long-lived species, such as conifers, but whose long generation times pose substantive challenges, including increased recalcitrance for se as plants age. identification of a clonal line of somatic embryo-derived trees whose shoot primordia have remained responsive to se induction for over a decade, provided a unique opportunity to examine the molecular aspects underpinning se within shoot tissues of adult white spruce trees.  microarray analysis was used to conduct transcriptome-wide expression profiling of shoot explants taken from this responsive genotype following one week of se induction, which when compared with that of a nonresponsive genotype, led to the identification of four of the most differentially expressed genes within each genotype. using absolute qpcr to expand the analysis to three weeks of induction revealed that differential expression of all eight candidate genes was maintained to the end of the induction treatment, albeit to differing degrees. most striking was that both the magnitude and duration of candidate gene expression within the nonresponsive genotype was indicative of an intense physiological response. examining their putative identities further revealed that all four encoded for proteins with similarity to angiosperm proteins known to play prominent roles in biotic defense, and that their high-level induction over an extended period is consistent with activation of a biotic defense response. in contrast, the more temperate response within the responsive genotype, including induction of a conifer-specific dehydrin, is more consistent with elicitation of an adaptive stress response.  while additional evidence is required to definitively establish an association between se responsiveness and a specific physiological response, these results suggest that biotic defense activation may be antagonistic, likely related to the massive transcriptional and metabolic reprogramming that it elicits. a major issue for future work will be to determine how and if suppressing biotic defense activation could be used to promote a physiological state more conducive to se induction. conifergene expression profilingmicroarray analysisabsolute qpcrlre qpcrclonal propagation 