Gene expression programs during callus development in tissue culture of two Eucalyptus species

Background Eucalyptus is a highly diverse genus of the Myrtaceae family and widely planted in the world for timber and pulp production. Tissue culture induced callus has become a common tool for Eucalyptus breeding, however, our knowledge about the genes related to the callus maturation and shoot regeneration is still poor. Results We set up an experiment to monitor the callus induction and callus development of two Eucalyptus species - E. camaldulensis (high embryogenic potential) and E. grandis x urophylla (low embryogenic potential). Then, we performed transcriptome sequencing for primary callus, mature callus, shoot regeneration stage callus and senescence callus. We identified 707 upregulated and 694 downregulated genes during the maturation process of the two Eucalyptus species and most of them were involved in the signaling pathways like plant hormone and MAPK. Next, we identified 135 and 142 genes that might play important roles during the callus development of E. camaldulensis and E. grandis x urophylla, respectively. Further, we found 15 DEGs shared by these two Eucalyptus species during the callus development, including Eucgr.D00640 (stem-specific protein TSJT1), Eucgr.B00171 (BTB/POZ and TAZ domain-containing protein 1), Eucgr.C00948 (zinc finger CCCH domain-containing protein 20), Eucgr.K01667 (stomatal closure-related actinbinding protein 3), Eucgr.C00663 (glutaredoxin-C10) and Eucgr.C00419 (UPF0481 protein At3g47200). Interestingly, the expression patterns of these genes displayed “N” shape in the samples. Further, we found 51 genes that were dysregulated during the callus development of E. camaldulensis but without changes in E. grandis x urophylla, such as Eucgr.B02127 (GRF1-interacting factor 1), Eucgr.C00947 (transcription factor MYB36), Eucgr.B02752 (laccase-7), Eucgr.B03985 (transcription factor MYB108), Eucgr.D00536 (GDSL esterase/lipase At5g45920) and Eucgr.B02347 (scarecrow-like protein 34). These 51 genes might be associated with the high propagation ability of Eucalyptus and 22 might be induced after the dedifferentiation. Last, we performed WGCNA to identify the co-expressed genes during the callus development of Eucalyptus and qRT-PCR experiment to validate the gene expression patterns. Conclusions This is the first time to globally study the gene profiles during the callus development of Eucalyptus. The results will improve our understanding of gene regulation and molecular mechanisms in the callus maturation and shoot regeneration.

Genotype and transcriptome effects on somatic embryogenesis in Cryptomeria japonica

Somatic embryogenesis (SE), which is in vitro regeneration of plant bodies from somatic cells, represents a useful means of clonal propagation and genetic engineering of forest trees. While protocols to obtain calluses and induce regeneration in somatic embryos have been reported for many tree species, the knowledge of molecular mechanisms of SE development is still insufficient to achieve an efficient supply of somatic embryos required for the industrial application. Cryptomeria japonica, a conifer species widely used for plantation forestry in Japan, is one of the tree species waiting for a secure SE protocol; the probability of normal embryo development appears to depend on genotype. To discriminate the embryogenic potential of embryonal masses (EMs) and efficiently obtain normal somatic embryos of C. japonica, we investigated the effects of genotype and transcriptome on the variation in embryogenic potential. Using an induction experiment with 12 EMs each from six genotypes, we showed that embryogenic potential differs between/within genotypes. Comparisons of gene expression profiles among EMs with different embryogenic potentials revealed that 742 differently expressed genes were mainly associated with pattern forming and metabolism. Thus, we suggest that not only genotype but also gene expression profiles can determine success in SE development. Consistent with previous findings for other conifer species, genes encoding leafy cotyledon, wuschel, germin-like proteins, and glutathione-S-transferases are likely to be involved in SE development in C. japonica and indeed highly expressed in EMs with high-embryogenic potential; therefore, these proteins represent candidate markers for distinguishing embryogenic potential.

Polycomb Repressive Complex 2-mediated histone modification H3K27me3 is associated with embryogenic potential in Norway spruce

Epigenetic reprogramming during germ cell formation is essential to gain pluripotency and thus embryogenic potential. The histone modification H3K27me3, which is catalysed by the Polycomb repressive complex 2 (PRC2), regulates important developmental processes in both plants and animals, and defects in PRC2 components cause pleiotropic developmental abnormalities. Nevertheless, the role of H3K27me3 in determining embryogenic potential in gymnosperms is still elusive. To address this, we generated H3K27me3 profiles of Norway spruce (Picea abies) embryonic callus and non-embryogenic callus using CUT&RUN, which is a powerful method for chromatin profiling. Here, we show that H3K27me3 mainly accumulated in genic regions in the Norway spruce genome, similarly to what is observed in other plant species. Interestingly, H3K27me3 levels in embryonic callus were much lower than those in the other examined tissues, but markedly increased upon embryo induction. These results show that H3K27me3 levels are associated with the embryogenic potential of a given tissue, and that the early phase of somatic embryogenesis is accompanied by changes in H3K27me3 levels. Thus, our study provides novel insights into the role of this epigenetic mark in spruce embryogenesis and reinforces the importance of PRC2 as a key regulator of cell fate determination across different plant species.

Tissue Culture Applications of Artemisia annua L. Callus for Indirect Organogenesis and Production Phytochemical

Artemisia annua L. axenic plants were used to induce callogeneisis for production of phenolics and plant regeneration. Up to 95% callogenesis from leaf and stem explants on MS supplemented with 2.0 mg/l NAA or 2,4-D + 0.2 mg/l BAP (MSC1 and MSC2) was observed. Lower callus frequency but with improved embryogenic potential was observed upon subculture on medium with reduced auxin and increased BAP concentration (0.5 mg/l NAA or 2,4-D + 0.5 mg/l BAP) (MSC3 and MSC4). The leafinduced callus on NAA/BAP (MSC3) showed best antioxidant potential. Induced shoot regeneration occurred upon high concentration BAP combined with NAA rather than with 2,4-D (0.25 mg/l NAA or 2,4-D + 1.0 mg/l BAP, MSR 1 and MSR2, respectively). Optimal shoot multiplication and rooting were obtained on 1.0 mg/l BAP and 0.1 mg/l IBA, respectively, followed by acclimatization of regenerants to greenhouse conditions. This work aims at establishing protocol for A. annua preservation and biosynthesis of natural products. Plant Tissue Cult. & Biotech. 30(1): 97-106, 2020 (June)

The commitment of barley microspores into embryogenesis involves miRNA-directed regulation of members of the SPL, GRF and HD-ZIPIII transcription factor families

SUMMARYMicrospore embryogenesis is a model for developmental plasticity and cell fate decisions. To investigate the role of miRNAs in this development, we sequenced sRNAs and the degradome of barley microspores collected prior to (day 0) and after (days 2 and 5) the application of a stress treatment known to induce embryogenesis. Microspores isolated at these timepoints were uniform in both appearance and in their complements of sRNAs. We detected 68 miRNAs in microspores. The abundance of 51 of these miRNAs differed significantly during microspore development. One group of miRNAs was induced when the stress treatment was applied, prior to being repressed when microspores transitioned to embryogenesis. Another group of miRNAs were up-regulated in day-2 microspores and their abundance remained stable or increased in day-5 microspores, a timepoint at which the first clear indications of the transition towards embryogenesis were visible. Collectively, these miRNAs might play a role in the modulation of the stress response, the repression of gametic development, and/or the gain of embryogenic potential. A degradome analysis allowed us to validate the role of miRNAs in regulating 41 specific transcripts. We showed that the transition of microspores toward the embryogenesis pathway involves miRNA-directed regulation of members of the ARF, SPL, GRF and HD-ZIPIII transcription factor families. We noted that 41.5% of these targets were shared between day-2 and day-5 microspores while 26.8% were unique to day-5 microspores. The former set may act to disrupt transcripts involved in pollen development while the latter set may drive the commitment to embryogenesis.

In Vitro Tissue Culture in Brachypodium: Applications and Challenges

Brachypodium distachyon has become an excellent model for plant breeding and bioenergy grasses that permits many fundamental questions in grass biology to be addressed. One of the constraints to performing research in many grasses has been the difficulty with which they can be genetically transformed and the generally low frequency of such transformations. In this review, we discuss the contribution that transformation techniques have made in Brachypodium biology as well as how Brachypodium could be used to determine the factors that might contribute to transformation efficiency. In particular, we highlight the latest research on the mechanisms that govern the gradual loss of embryogenic potential in a tissue culture and propose using B. distachyon as a model for other recalcitrant monocots.

In vitro callus induction and development of Vernonia condensata Baker with embryogenic potential

ABSTRACT Vernonia condensata Baker has been traditionally used in folk medicine for the treatment of several inflammatory and infectious processes. Overexploitation of this plant species has drastically reduced its population in its natural habitat (Cerrado). Therefore, tissue culture tools, such as somatic embryogenesis, can be used as an alternative method for rapid and large-scale plant regeneration. The objectives of this study were to induce callogenesis in Vernonia condensata from different types of explants and to evaluate the structural aspects of the development of pro-embryogenic masses of this species by means of histological analyses. The formation of calli was induced from leaf explants and internodal segments, which were inoculated in EME medium supplemented with 50 g L-1 sucrose, 0.5 g L-1 malt extract and 2.68 μM NAA, plus varying concentrations of BAP (0.00, 2.22, 4.44 or 8.88 μM). After 40 days, the following morphogenetic traits were evaluated: intensity of callus formation, intensity of oxidation, callus texture, and morphogenesis. The calli with embryogenic masses were analyzed by light and scanning electron microscopy. Both types of explants were responsive regarding callogenesis, with the BAP concentration of 4.44 μM promoting the formation of friable calli associated with a larger percentage of calli with embryogenic masses. Cells from leaf explants and internodal segments were able to dedifferentiate and change into embryonic structures.

Embryogenic potential of the callus of gabirobeira, Campomanesia adamantium (Cambess) O. Berg

Campomanesia adamantium is a native plant species of Brazilian Cerrado with diverse economic potential and great medicinal importance. Its sexual propagation is impaired by the recalcitrance of its seeds, which prevents effective and profitable propagation. With the purpose of establishing commercial crops and minimizing the extractive use of vegetal resources, the aim of the present study was to induce embryogenic calli in nodal segments of gabirobeira, and to determine and characterize their embryogenic phase through the establishment of a growth curve based on cellular characteristics. Calli were induced using nodal segments inoculated in WPM culture medium without the addition of hormones (control) and with different concentrations of 2,4-D, IAA, IBA, NAA or picloram. Cytochemical and SEM analyses revealed cellular characteristics of the formation of meristematic centers that indicated 4.14 μM of picloram to be the best treatment for induction of embryogenic calli, and demonstrating their embryogenic potential. The treatment was used to establish a callus growth curve, from which it was inferred that calli should be transferred to new culture media on the 28th day to maintain cell viability