David I. Dunstan

Improved high-level constitutive foreign gene expression in plants using an AMV RNA4 untranslated leader sequence

Abstract A synthetic transcribed, untranslated leader sequence from alfalfa mosaic virus RNA4 (AMV leader) has been assessed for its in vivo properties as a cis -active ‘translational activator’ in transient expression assays in protoplasts of Nicotiana tabacum and Picea glauca , as well as in stable expression in transformed Nicotiana tabacum . Levels of GUS enzyme activity produced by chimeric genes with or without the AMV leader sequence, in combination with either a CaMV 35S promoter or a duplicated-enhancer CaMV 35S promoter construct were assessed. In transient assay systems, the presence of a synthetic 40-base leader sequence lead to a 20-fold elevation in GUS activity when the constructs contained a native cauliflower mosaic virus (CaMV) 35S promoter, whereas a 4-fold elevated expression lebel was observed in constructs containing a duplicated-enhancer 35S promoter. Furthermore, elevated expression in chimeric constructs was influenced by the sequence context for translation initiation of the marker gene. In transgenic tobacco plants the mean values for steady-state expression of GUS-containing 35S/AMV constructs were elevated about 8-fold relative to plasmids containing the native 35S promoter alone. A quantitative PCR approach was used to assess relative transcript levels in plants expressing GUS from AMV-containing chimeric constructs. The results showed that elevated expression attributable to the AMV leader sequence was independent of abundance of the corresponding AMV- gus transcript, suggesting a post-transcriptional mechanism of action in vivo. Further, we describe the construction of general-purpose constitutive high expression plant promoter cassettes which incorporate the AMV translational enhancer sequence, as well a duplicated-enhancer 35S promoter in an optimized translational context.

Effects of abscisic acid and analogues on the maturation of white spruce (Picea glauca) somatic embryos

Abstract Somatic embryo-competent cultures of white spruce, Picea glauca (Moench) Voss, were grown on (±)-abscisic acid (ABA) and three analogues known to be biotransformed by intact plants into ABA: (±)-methyl abscisate (MeABA); (±)-ethyl-(2E,4E) and -(2E,4Z)-5-(1′,2′-epoxy-2′,6′,6′-trimethylcyclohexyl)-3-methylpentadienoate (epoxyester, EE); and (±)-(2E,4E) and -(2E,4Z)-5-(1′,2′-epoxy-2′,6′,6′-trimethylcyclohexyl)-3-methylpentadienocic acid (epoxyacid, EA). ABA between 8 and 12 μM most efficiently promoted embryo maturation and led to the maximum recovery (4%) of stage 4b embryos when used in the absence of auxin or cytokinin. MeABA in the absence of auxin and cytokinin also promoted embryo maturation, EE promoted only the early stages in maturation, and EA was unable to promote any maturation of somatic embryos. Phytohormone-free medium produced very similar responses to EA, though in rare cases stage 3 embryos were found. Neither ABA nor the analogues promoted embryo maturation when used in combination with 9 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 μM N6-benzyladenine (BA).

Endochitinase and β-1,3-glucanase genes are developmentally regulated during somatic embryogenesis inPicea glauca

Two cDNAs isolated from white spruce [Picea glauca (Moench) Voss] somatic embryos, are predicted to encode a basic class IV chitinase and a β-1,3-glucanase, respectively corresponding to genesPgChi-1 andPgGlu-1. Each represents a multigene family in spruce. Transcripts homologous toPgChi-1 orPgGlu-1 genes were highly abundant in embryogenic tissues and gradually decreased after tissues were placed on abscisic acid-containing maturation medium, with lowest abundance in globular embryos. Transcripts related toPgGlu-1 became highly abundant again in early cotyledonary embryos but decreased thereafter, whereas transcripts related toPgChi-1 were also highly abundant in late cotyledonary embryos and plantlets in vitro; transcripts were either low (PgChi-1) or were not detectable (PgGlu-1) in needles. Wounding, drying and flooding stresses enhancedPgChi-1-andPgGlu-1-related gene expression. Fungal cell wall suspension enhancedPgGlu-1-related transcript accumulation, but reducedPgChi-1-related transcript abundance within 24 h.PgChi-1 andPgGlu-1 and their homologues may have roles in plant defense, and possibly developmental roles during spruce somatic embryo maturation.

Expression of abundant mRNAs during somatic embryogenesis of white spruce [Picea glauca (Moench) Voss].

Embryogenic tissues of white spruce [Picea glauca (Moench) Voss] remain in an early developmental stage while cultured on 2,4-dichlorophenoxyacetic acid and N6-benzyladenine, but develop to cotyledonary embryos when these phytohormones are replaced by abscisic acid. Twenty-eight cDNAs were isolated from cotyledonary embryos by differential screening against immature embryo and non-embryonic tissues. Temporal expression patterns of these cDNAs during ABA-stimulated somatic embryo development were observed. This showed that clones could be allocated to various groups, including embryo-abundant, embryo-maturation-induced, and those whose expression was modulated during embryo development, germination or in non-embryogenic tissues. Expression corresponding to these cDNA clones showed that there were various responses to exogenous ABA or polyethylene glycol during a period of 48 h. Analyses of DNA and predicted amino acid sequence revealed that 12 of 28 cDNA clones had no known homologues, while others were predicted to encode different late-embryogenesis-abundant proteins, early methionine-labelled proteins, storage proteins, heat-shock proteins, glycine-rich cell wall protein, metallothionein-like protein and some other metabolic enzymes.

Somatic embryogenesis in woody plants.

Woody plants may be defined as trees and shrubs “whose stems live for a number of years and increase in diameter each year by addition of woody tissue” [1]. This contrasts with the description for herbaceous plants “with a relatively short-lived aboveground stem that is comparatively thin, soft, and nonwoody” [1]. Included in this definition of woody plants are those vines which are woody, i.e. “plants whose stems are not self-supporting, but either trail on the ground or climb by attaching to other plants or supports” [1]. In this context species such as Manihot esculenta and Carica papaya, included as woody by others [2,3] are regarded as herbaceous here.

Metabolism of (+)- and (−)-abscisic acid by somatic embryo suspension cultures of white spruce

Abstract Somatic embryo suspension cultures of white spruce in medium containing (+)-abscisic acid [(+)-ABA], at an initial concentration of 15 μM, metabolized the ABA essentially completely within seven days. The metabolites accumulated in the liquid medium. The (+)-ABA was converted almost quantitively to phaseic acid, with little further transformation into dihydrophaseic acid. The sum of the concentrations of the two metabolites in the medium closely approximated the concentration of (+)-ABA initially supplied. (−)-ABA remained essentially unchanged under the same culture conditions, and when the cells were supplied with racemic (±)-ABA, only the (+) enantiomer was metabolized. When (+)- or (±)-ABA was present in the medium, the size of the embryos and their suspensors increased during the culture period, a development consistent with somatic embryo maturation. Embryos in control cultures lacking exogenously supplied ABA, and embryos in cultures provided with only (−)-ABA, did not increase in size. The early disappearance of the (+)-ABA from the suspension medium, and the completeness of its conversion to phaseic acid, raises the question of the relative roles of ABA and phaseic acid in the maturation of conifer somatic embryos.

Cryopreservation of interior spruce (Picea glauca engelmanni complex) embryogenic cultures

SummaryEmbryogenic cultures of interior spruce derived from 12 full-sib families were subjected to cryopreservation, with a 97 % success rate for 357 genotypes. Analyses suggested that cryotolerance was not related to family ranking (height increment), embryogenic potential or culture dispersability in suspension, and long-term storage in or above liquid nitrogen did not affect regenerative potential. By contrast, differences in cryotolerance among cell lines appeared to be prevalent in certain families. Analysis with a DNA fingerprinting probe used for clonal identification demonstrated no evidence of somaclonal variation as a result of cryopreservation. The results of this work indicate the applicability of cryopreservation as a long-term storage strategy for spruce embryogenic cultures from a wide genetic background.

Somatic embryogenesis from immature and mature zygotic embryos, and embryo regeneration from protoplasts in black spruce (Picea mariana Mill.).

Abstract Embryogenic calli of black spruce (Picea mariana Mill.) were initiated in vitro from immature zygotic embryos collected during the maturation period, and with mature zygotic embryos dissected from seed which had been stored for 13 years. With immature zygotic embryos as explants, highest frequency of embryogenic calli (65%) was obtained using full strength von Arnold and Eriksson (LP) medium in the absence of casein hydrolysate (CH). With mature zygotic embryos, embryogenic calli developed in 8% of explants on this medium and in 10% of explants using 1 2 strength modified Litvay ( 1 2 LM ) medium. Calli derived from mature embryos were grown as suspensions in liquid medium. Protoplasts isolated from six of these suspension lines differed in their response to culture conditions. In 1 2 LM (liquid, or agarose solidified) with 0.37 M glucose as osmoticum, protoplasts of two lines showed first divisions after 24 h and recognizable somatic embryos had developed by 7 days. Protoplasts from the other lines either failed during their development into somatic embryos, or developed into green non-embryogenic callus.

Regeneration of somatic embryos from protoplasts isolated from an embryogenic suspension culture of white spruce (Picea glauca)

Regeneration of white spruce (Picea glauca) somatic embryos from protoplasts derived from an embryogenic suspension culture was accomplished using a culture medium containing 2 mgl−1 2,4-D and 1 mgl−1 6-BAP. Divisions within 2 days led to plating efficiencies in the order of 24% after 9 days. A reduction in the osmoticum, necessary for sustained growth, was carried out gradually over 30 days. Embedding in agarose and culture in 5 cm petri dishes prior to transfer of agarose blocks to a bead type culture, led to the formation of somatic embryos as early as 23 days after isolation and yielded plating efficiencies in the order of 5–10% after 35 days culture.

Racemic abscisic acid and abscisyl alcohol promote maturation of white spruce (Picea glauca) somatic embryos

Abstract Racemic abscisic acid [(±)-ABA] and racemic abscisyl alcohol were used to obtain maturation of white spruce (Picea glauca (Moench.) Voss) immature (stage 1) somatic embryos. Data for somatic embryo development were collected between 21 and 70 days after initiation of maturation treatments. The culture period for the maximum production of cotyledonary (stage 3) somatic embryos was approx. 42 days, with either 46 μM (±)-ABA or 41 μM abscisyl alcohol during the first 35 days. Two additional abscisic acid analogues, an acetylenic aldehyde and a dihydroacetylenic alcohol were less effective in promoting somatic embryo maturation. A comparison of the molecular structures of compounds indicated that the presence of an ABA-like side chain with two double bonds was most effective in white spruce somatic embryo maturation. Another factor that influenced the maturation process was the solvent used to dissolve (±)-ABA and the analogues. Somatic embryo development could be arrested at stage 3 by extending exposure to 60 μM (±)-ABA for up to 9 weeks. Germination resulted after removal of (±)-ABA.