Chromosome-Scale Genome Assembly of the Sheep-Biting Louse Bovicola ovis Using Nanopore Sequencing Data and Pore-C Analysis.

Bovicola ovis, commonly known as the sheep-biting louse, is an ectoparasite that adversely affects the sheep industry. Sheep louse infestation lowers the quality of products, including wool and leather, causing a loss of approximately AUD 123M per annum in Australia alone. The lack of a high-quality genome assembly for the sheep-biting louse, as well as any closely related livestock lice, has hindered the development of louse research and management control tools. In this study, we present the assembly of B. ovis with a genome size of ~123 Mbp based on a nanopore long-read sequencing library and Illumina RNA sequencing, complemented with a chromosome-level scaffolding using the Pore-C multiway chromatin contact dataset. Combining multiple alignment and gene prediction tools, a comprehensive annotation on the assembled B. ovis genome was conducted and recalled 11,810 genes as well as other genomic features including orf, ssr, rRNA and tRNA. A manual curation using alignment with the available closely related louse species, Pediculus humanus, increased the number of annotated genes to 16,024. Overall, this study reported critical genetic resources and biological insights for the advancement of sheep louse research and the development of sustainable control strategies in the sheep industry.

Clay nanoparticles efficiently deliver small interfering RNA to intact plant leaf cells.

RNA interference is triggered in plants by the exogenous application of double-stranded RNA or small interfering RNA (siRNA) to silence the expression of target genes. This approach can potentially provide insights into metabolic pathways and gene function and afford plant protection against viruses and other plant pathogens. However, the effective delivery of biomolecules such as siRNA into plant cells is difficult because of the unique barrier imposed by the plant cell wall. Here, we demonstrate that 40-nm layered double hydroxide (LDH) nanoparticles are rapidly taken up by intact Nicotiana benthamiana leaf cells and by chloroplasts, following their application via infiltration. We also describe the distribution of infiltrated LDH nanoparticles in leaves and demonstrate their translocation through the apoplast and vasculature system. Furthermore, we show that 40-nm LDH nanoparticles can greatly enhance the internalization of nucleic acids by N. benthamiana leaf cells to facilitate siRNA-mediated downregulation of targeted transgene mRNA by >70% within 1 day of exogenous application. Together, our results show that 40-nm LDH nanoparticle is an effective platform for delivery of siRNA into intact plant leaf cells.

Synergistic Effect of Two Nanotechnologies Enhances the Protective Capacity of the Theileria parva Sporozoite p67C Antigen in Cattle.

East Coast fever (ECF), caused by Theileria parva, is the most important tick-borne disease of cattle in sub-Saharan Africa. Practical disadvantages associated with the currently used live-parasite vaccine could be overcome by subunit vaccines. An 80-aa polypeptide derived from the C-terminal portion of p67, a sporozoite surface Ag and target of neutralizing Abs, was the focus of the efforts on subunit vaccines against ECF and subjected to several vaccine trials with very promising results. However, the vaccination regimen was far from optimized, involving three inoculations of 450 µg of soluble p67C (s-p67C) Ag formulated in the Seppic adjuvant Montanide ISA 206 VG. Hence, an improved formulation of this polypeptide Ag is needed. In this study, we report on two nanotechnologies that enhance the bovine immune responses to p67C. Individually, HBcAg-p67C (chimeric hepatitis B core Ag virus-like particles displaying p67C) and silica vesicle (SV)-p67C (s-p67C adsorbed to SV-140-C18, octadecyl-modified SVs) adjuvanted with ISA 206 VG primed strong Ab and T cell responses to p67C in cattle, respectively. Coimmunization of cattle (Bos taurus) with HBcAg-p67C and SV-p67C resulted in stimulation of both high Ab titers and CD4 T cell response to p67C, leading to the highest subunit vaccine efficacy we have achieved to date with the p67C immunogen. These results offer the much-needed research depth on the innovative platforms for developing effective novel protein-based bovine vaccines to further the advancement.

RNA-Based Control of Fungal Pathogens in Plants.

Our duty to conserve global natural ecosystems is increasingly in conflict with our need to feed an expanding population. The use of conventional pesticides not only damages the environment and vulnerable biodiversity but can also still fail to prevent crop losses of 20-40% due to pests and pathogens. There is a growing call for more ecologically sustainable pathogen control measures. RNA-based biopesticides offer an eco-friendly alternative to the use of conventional fungicides for crop protection. The genetic modification (GM) of crops remains controversial in many countries, though expression of transgenes inducing pathogen-specific RNA interference (RNAi) has been proven effective against many agronomically important fungal pathogens. The topical application of pathogen-specific RNAi-inducing sprays is a more responsive, GM-free approach to conventional RNAi transgene-based crop protection. The specific targeting of essential pathogen genes, the development of RNAi-nanoparticle carrier spray formulations, and the possible structural modifications to the RNA molecules themselves are crucial to the success of this novel technology. Here, we outline the current understanding of gene silencing pathways in plants and fungi and summarize the pioneering and recent work exploring RNA-based biopesticides for crop protection against fungal pathogens, with a focus on spray-induced gene silencing (SIGS). Further, we discuss factors that could affect the success of RNA-based control strategies, including RNA uptake, stability, amplification, and movement within and between the plant host and pathogen, as well as the cost and design of RNA pesticides.

Sheet-like clay nanoparticles deliver RNA into developing pollen to efficiently silence a target gene.

Topical application of double-stranded RNA (dsRNA) can induce RNA interference (RNAi) and modify traits in plants without genetic modification. However, delivering dsRNA into plant cells remains challenging. Using developing tomato (Solanum lycopersicum) pollen as a model plant cell system, we demonstrate that layered double hydroxide (LDH) nanoparticles up to 50 nm in diameter are readily internalized, particularly by early bicellular pollen, in both energy-dependent and energy-independent manners and without physical or chemical aids. More importantly, these LDH nanoparticles efficiently deliver dsRNA into tomato pollen within 2-4 h of incubation, resulting in an 89% decrease in transgene reporter mRNA levels in early bicellular pollen 3-d post-treatment, compared with a 37% decrease induced by the same dose of naked dsRNA. The target gene silencing is dependent on the LDH particle size, the dsRNA dose, the LDH-dsRNA complexing ratio, and the treatment time. Our findings indicate that LDH nanoparticles are an effective nonviral vector for the effective delivery of dsRNA and other biomolecules into plant cells.

Temporal expression of defence and susceptibility genes and tospovirus accumulation in capsicum chlorosis virus-infected capsicum.

Yolo Wonder (YW) and Warlock (W), two capsicum cultivars that are susceptible to capsicum chlorosis virus (CaCV), were compared in terms of symptom development, tospovirus accumulation, and host gene expression during the first 12 days post infection (dpi). Temporal expression of selected early CaCV-response genes was used to gain insights into plant-virus interactions and to identify potential targets for CaCV control. Symptoms developed faster in YW during the first seven days of infection, while systemic symptoms were similar in both cultivars at 10 and 12 dpi. CaCV accumulation was higher in YW at 7 dpi despite a lower titre at 3 dpi. At 12 dpi, virus accumulation was similar for both cultivars. Symptom development appears to be correlated to virus accumulation over time for both cultivars. Chalcone synthase (CHS), cytochrome P450 (CYP), and tetraspanin 8-like (TSP8) genes followed a similar expression pattern over time in both cultivars. The thionin gene showed increased expression in CaCV-infected plants at 12 dpi. The WRKY40 gene showed significant differential expression at all time points in YW, but only at 12 dpi in W. The strongest correlation of temporal gene expression and virus titre was seen for CYP, TSP8, thionin, and WRKY40. CHS and CYP may be involved in symptom development, and TSP8 may be involved in virus movement. CHS, CYP, and TSP8 may be good targets for future overexpression or silencing studies to clarify their functions during virus infection and, potentially, for control of CaCV in capsicum.

The avocado genome informs deep angiosperm phylogeny, highlights introgressive hybridization, and reveals pathogen-influenced gene space adaptation.

The avocado, Persea americana, is a fruit crop of immense importance to Mexican agriculture with an increasing demand worldwide. Avocado lies in the anciently diverged magnoliid clade of angiosperms, which has a controversial phylogenetic position relative to eudicots and monocots. We sequenced the nuclear genomes of the Mexican avocado race, P. americana var. drymifolia, and the most commercially popular hybrid cultivar, Hass, and anchored the latter to chromosomes using a genetic map. Resequencing of Guatemalan and West Indian varieties revealed that ∼39% of the Hass genome represents Guatemalan source regions introgressed into a Mexican race background. Some introgressed blocks are extremely large, consistent with the recent origin of the cultivar. The avocado lineage experienced 2 lineage-specific polyploidy events during its evolutionary history. Although gene-tree/species-tree phylogenomic results are inconclusive, syntenic ortholog distances to other species place avocado as sister to the enormous monocot and eudicot lineages combined. Duplicate genes descending from polyploidy augmented the transcription factor diversity of avocado, while tandem duplicates enhanced the secondary metabolism of the species. Phenylpropanoid biosynthesis, known to be elicited by Colletotrichum (anthracnose) pathogen infection in avocado, is one enriched function among tandems. Furthermore, transcriptome data show that tandem duplicates are significantly up- and down-regulated in response to anthracnose infection, whereas polyploid duplicates are not, supporting the general view that collections of tandem duplicates contribute evolutionarily recent "tuning knobs" in the genome adaptive landscapes of given species.

RNAi as a Foliar Spray: Efficiency and Challenges to Field Applications.

RNA interference (RNAi) is a powerful tool that is being increasingly utilized for crop protection against viruses, fungal pathogens, and insect pests. The non-transgenic approach of spray-induced gene silencing (SIGS), which relies on spray application of double-stranded RNA (dsRNA) to induce RNAi, has come to prominence due to its safety and environmental benefits in addition to its wide host range and high target specificity. However, along with promising results in recent studies, several factors limiting SIGS RNAi efficiency have been recognized in insects and plants. While sprayed dsRNA on the plant surface can produce a robust RNAi response in some chewing insects, plant uptake and systemic movement of dsRNA is required for delivery to many other target organisms. For example, pests such as sucking insects require the presence of dsRNA in vascular tissues, while many fungal pathogens are predominately located in internal plant tissues. Investigating the mechanisms by which sprayed dsRNA enters and moves through plant tissues and understanding the barriers that may hinder this process are essential for developing efficient ways to deliver dsRNA into plant systems. In this review, we assess current knowledge of the plant foliar and cellular uptake of dsRNA molecules. We will also identify major barriers to uptake, including leaf morphological features as well as environmental factors, and address methods to overcome these barriers.

BioClay™ prolongs RNA interference-mediated crop protection against Botrytis cinerea.

One of the most promising tools for the control of fungal plant diseases is spray-induced gene silencing (SIGS). In SIGS, small interfering RNA (siRNA) or double-stranded RNA (dsRNA) targeting essential or virulence-related pathogen genes are exogenously applied to plants and postharvest products to trigger RNA interference (RNAi) of the targeted genes, inhibiting fungal growth and disease. However, SIGS is limited by the unstable nature of RNA under environmental conditions. The use of layered double hydroxide or clay particles as carriers to deliver biologically active dsRNA, a formulation termed BioClay™, can enhance RNA durability on plants, prolonging its activity against pathogens. Here, we demonstrate that dsRNA delivered as BioClay can prolong protection against Botrytis cinerea, a major plant fungal pathogen, on tomato leaves and fruit and on mature chickpea plants. BioClay increased the protection window from 1 to 3 weeks on tomato leaves and from 5 to 10 days on tomato fruits, when compared with naked dsRNA. In flowering chickpea plants, BioClay provided prolonged protection for up to 4 weeks, covering the critical period of poding, whereas naked dsRNA provided limited protection. This research represents a major step forward for the adoption of SIGS as an eco-friendly alternative to traditional fungicides.

Scion control of miRNA abundance and tree maturity in grafted avocado.

BACKGROUND: Grafting is the common propagation method for avocado and primarily benefits orchard production by reducing the time to tree productivity. It also allows use of scions and rootstocks specifically selected for improved productivity and commercial acceptance. Rootstocks in avocado may be propagated from mature tree cuttings ('mature'), or from seed ('juvenile'). While the use of mature scion material hastens early bearing/maturity and economic return, the molecular factors involved in the role of the scion and/or rootstock in early bearing/reduced juvenility of the grafted tree are still unknown. RESULTS: Here, we utilized juvenility and flowering associated miRNAs; miR156 and miR172 and their putative target genes to screen pre-graft and post-graft material in different combinations from avocado. The abundance of mature miR156, miR172 and the miR156 target gene SPL4, showed a strong correlation to the maturity of the scion and rootstock material in avocado. Graft transmissibility of miR156 and miR172 has been explored in annual plants. Here, we show that the scion may be responsible for grafted tree maturity involving these factors, while the rootstock maturity does not significantly influence miRNA abundance in the scion. We also demonstrate that the presence of leaves on cutting rootstocks supports graft success and contributes towards intergraft signalling involving the carbohydrate-marker TPS1. CONCLUSION: Here, we suggest that the scion largely controls the molecular 'maturity' of grafted avocado trees, however, leaves on the rootstock not only promote graft success, but can influence miRNA and mRNA abundance in the scion. This constitutes the first study on scion and rootstock contribution towards grafted tree maturity using the miR156-SPL4-miR172 regulatory module as a marker for juvenility and reproductive competence.

SCRAM: a pipeline for fast index-free small RNA read alignment and visualization.

Summary: Small RNAs play key roles in gene regulation, defense against viral pathogens and maintenance of genome stability, though many aspects of their biogenesis and function remain to be elucidated. SCRAM (Small Complementary RNA Mapper) is a novel, simple-to-use short read aligner and visualization suite that enhances exploration of small RNA datasets. Availability and implementation: The SCRAM pipeline is implemented in Go and Python, and is freely available under MIT license. Source code, multiplatform binaries and a Docker image can be accessed via https://sfletc.github.io/scram/. Supplementary information: Supplementary data are available at Bioinformatics online.

Molecular characterization and analysis of conserved potyviral motifs in bean common mosaic virus (BCMV) for RNAi-mediated protection.

Australian bean common mosaic virus (BCMV) isolates were sequenced, and the sequences were compared to global BCMV and bean common mosaic necrosis virus (BCMNV) sequences and analysed for conserved potyviral motifs to generate in planta RNA-interference (RNAi) resistance. Thirty-nine out of 40 previously reported potyvirus motifs were conserved among all 77 BCMV/BCMNV sequences. Two RNAi target regions were selected for dsRNA construct design, covering 920 bp of the nuclease inclusion b (NIb) protein and 461 bp of the coat protein (CP). In silico prediction of the effectiveness of these constructs for broad-spectrum defence against the 77 BCMV and BCMNV sequences was done via analysis of putative 21-nucleotide (nt) and 22-nt small-interfering RNAs (siRNAs) generated from the target regions. The effectiveness of both constructs for siRNA generation and BCMV RNAi-mediated resistance was validated in Nicotiana benthamiana transient assays.

A Genetic Screen for Impaired Systemic RNAi Highlights the Crucial Role of DICER-LIKE 2.

Posttranscriptional gene silencing (PTGS) of transgenes involves abundant 21-nucleotide small interfering RNAs (siRNAs) and low-abundance 22-nucleotide siRNAs produced from double-stranded RNA (dsRNA) by DCL4 and DCL2, respectively. However, DCL2 facilitates the recruitment of RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) to ARGONAUTE 1-derived cleavage products, resulting in more efficient amplification of secondary and transitive dsRNA and siRNAs. Here, we describe a reporter system where RDR6-dependent PTGS is initiated by restricted expression of an inverted-repeat dsRNA specifically in the Arabidopsis (Arabidopsis thaliana) root tip, allowing a genetic screen to identify mutants impaired in RDR6-dependent systemic PTGS. Our screen identified dcl2 but not dcl4 mutants. Moreover, grafting experiments showed that DCL2, but not DCL4, is required in both the source rootstock and the recipient shoot tissue for efficient RDR6-dependent systemic PTGS. Furthermore, dcl4 rootstocks produced more DCL2-dependent 22-nucleotide siRNAs than the wild type and showed enhanced systemic movement of PTGS to grafted shoots. Thus, along with its role in recruiting RDR6 for further amplification of PTGS, DCL2 is crucial for RDR6-dependent systemic PTGS.

The effects of potato virus Y-derived virus small interfering RNAs of three biologically distinct strains on potato (Solanum tuberosum) transcriptome.

BACKGROUND: Potato virus Y (PVY) is one of the most economically important pathogen of potato that is present as biologically distinct strains. The virus-derived small interfering RNAs (vsiRNAs) from potato cv. Russet Burbank individually infected with PVY-N, PVY-NTN and PVY-O strains were recently characterized. Plant defense RNA-silencing mechanisms deployed against viruses produce vsiRNAs to degrade homologous viral transcripts. Based on sequence complementarity, the vsiRNAs can potentially degrade host RNA transcripts raising the prospect of vsiRNAs as pathogenicity determinants in virus-host interactions. This study investigated the global effects of PVY vsiRNAs on the host potato transcriptome. METHODS: The strain-specific vsiRNAs of PVY, expressed in high copy number, were analyzed in silico for their proclivity to target potato coding and non-coding RNAs using psRobot and psRNATarget algorithms. Functional annotation of target coding transcripts was carried out to predict physiological effects of the vsiRNAs on the potato cv. Russet Burbank. The downregulation of selected target coding transcripts was further validated using qRT-PCR. RESULTS: The vsiRNAs derived from biologically distinct strains of PVY displayed diversity in terms of absolute number, copy number and hotspots for siRNAs on their respective genomes. The vsiRNAs populations were derived with a high frequency from 6 K1, P1 and Hc-Pro for PVY-N, P1, Hc-Pro and P3 for PVY-NTN, and P1, 3' UTR and NIa for PVY-O genomic regions. The number of vsiRNAs that displayed interaction with potato coding transcripts and number of putative coding target transcripts were comparable between PVY-N and PVY-O, and were relatively higher for PVY-NTN. The most abundant target non-coding RNA transcripts for the strain specific PVY-derived vsiRNAs were found to be MIR821, 28S rRNA,18S rRNA, snoR71, tRNA-Met and U5. Functional annotation and qRT-PCR validation suggested that the vsiRNAs target genes involved in plant hormone signaling, genetic information processing, plant-pathogen interactions, plant defense and stress response processes in potato. CONCLUSIONS: The findings suggested that the PVY-derived vsiRNAs could act as a pathogenicity determinant and as a counter-defense strategy to host RNA silencing in PVY-potato interactions. The broad range of host genes targeted by PVY vsiRNAs in infected potato suggests a diverse role for vsiRNAs that includes suppression of host stress responses and developmental processes. The interactome scenario is the first report on the interaction between one of the most important Potyvirus genome-derived siRNAs and the potato transcripts.

Silica Nanopollens Enhance Adhesion for Long-Term Bacterial Inhibition.

Nature's creations with spiky topological features typically exhibit intriguing surface adhesive properties. From micrometer-sized pollen grains that can easily stick to hairy insects for pollination to nanoscale virus particles that are highly infectious toward host cells, multivalent interactions are formed taking advantage of rough surfaces. Herein, this nature-inspired concept is employed to develop novel drug delivery nanocarriers for antimicrobial applications. A facile new approach is developed to fabricate silica nanopollens (mesoporous silica nanospheres with rough surfaces), which show enhanced adhesion toward bacteria surfaces compared to their counterparts with smooth surfaces. Lysozyme, a natural antimicrobial enzyme, is loaded into silica nanopollens and shows sustained release behavior, potent antimicrobial activity, and long-term total bacterial inhibition up to 3 days toward Escherichia coli. The potent antibacterial activity of lysozyme-loaded silica nanopollens is further demonstrated ex vivo by using a small-intestine infection model. Our strategy provides a novel pathway in the rational design of nanocarriers for efficient drug delivery.

Genomic deletions and mutations resulting in the loss of eight genes reduce the in vivo replication capacity of Meleagrid herpesvirus 1.

Meleagrid herpesvirus 1 (MeHV-1 or turkey herpesvirus) has been widely used as a vaccine in commercial poultry. Initially, these vaccine applications were for the prevention of Marek's disease resulting from Gallid herpesvirus 2 infections, while more recently MeHV-1 has been used as recombinant vector for other poultry infections. The construction of herpesvirus infectious clones that permit propagation and manipulation of the viral genome in bacterial hosts has advanced the studies of herpesviral genetics. The current study reports the construction of five MeHV-1 infectious clones. The in vitro properties of viruses recovered from these clones were indistinguishable from the parental MeHV-1. In contrast, the rescued MeHV-1 viruses were significantly attenuated when used in vivo. Complete sequencing of the infectious clones identified the absence of two regions of the MeHV-1 genome compared to the MeHV-1 reference sequence. These analyses determined the rescued viruses have seven genes, UL43, UL44, UL45, UL56, HVT071, sorf3 and US2 either partially or completely deleted. In addition, single nucleotide polymorphisms were identified in all clones compared with the MeHV-1 reference sequence. As a consequence of one of the polymorphisms identified in the UL13 gene, four of the rescued viruses were predicted to encode a serine/threonine protein kinase lacking two of three domains required for activity. Thus four of the recovered viruses have a total of eight missing or defective genes. The implications of these findings in the context of herpesvirus biology and infectious clone construction are discussed.

Synthesis of silica vesicles with controlled entrance size for high loading, sustained release, and cellular delivery of therapeutical proteins.

A rationally designed two-step synthesis of silica vesicles is developed with the formation of vesicular structure in the first step and fine control over the entrance size by tuning the temperature in the second step. The silica vesicles have a uniform size of ≈50 nm with excellent cellular uptake performance. When the entrance size is equal to the wall thickness, silica vesicles after hydrophobic modification show the highest loading amount (563 mg/g) towards Ribonuclease A with a sustained release behavior. Consequently, the silica vesicles are excellent nano-carriers for cellular delivery applications of therapeutical biomolecules.

Mutational analysis of two highly conserved motifs in the silencing suppressor encoded by tomato spotted wilt virus (genus Tospovirus, family Bunyaviridae).

Tospoviruses cause serious economic losses to a wide range of field and horticultural crops on a global scale. The NSs gene encoded by tospoviruses acts as a suppressor of host plant defense. We identified amino acid motifs that are conserved in all of the NSs proteins of tospoviruses for which the sequence is known. Using tomato spotted wilt virus (TSWV) as a model, the role of these motifs in suppressor activity of NSs was investigated. Using site-directed point mutations in two conserved motifs, glycine, lysine and valine/threonine (GKV/T) at positions 181-183 and tyrosine and leucine (YL) at positions 412-413, and an assay to measure the reversal of gene silencing in Nicotiana benthamiana line 16c, we show that substitutions (K182 to A, and L413 to A) in these motifs abolished suppressor activity of the NSs protein, indicating that these two motifs are essential for the RNAi suppressor function of tospoviruses.

Involvement of MicroRNAs in the Hypersensitive Response of Capsicum Plants to the Capsicum Chlorosis Virus at Elevated Temperatures.

The orthotospovirus capsicum chlorosis virus (CaCV) is an important pathogen affecting capsicum plants. Elevated temperatures may affect disease progression and pose a potential challenge to capsicum production. To date, CaCV-resistant capsicum breeding lines have been established; however, the impact of an elevated temperature of 35 °C on this genetic resistance remains unexplored. Thus, this study aimed to investigate how high temperature (HT) influences the response of CaCV-resistant capsicum to the virus. Phenotypic analysis revealed a compromised resistance in capsicum plants grown at HT, with systemic necrotic spots appearing in 8 out of 14 CaCV-infected plants. Molecular analysis through next-generation sequencing identified 105 known and 83 novel microRNAs (miRNAs) in CaCV-resistant capsicum plants. Gene ontology revealed that phenylpropanoid and lignin metabolic processes, regulated by Can-miR408a and Can- miR397, are likely involved in elevated-temperature-mediated resistance-breaking responses. Additionally, real-time PCR validated an upregulation of Can-miR408a and Can-miR397 by CaCV infection at HT; however, only the Laccase 4 transcript, targeted by Can-miR397, showed a tendency of negative correlation with this miRNA. Overall, this study provides the first molecular insights into how elevated temperature affects CaCV resistance in capsicum plants and reveals the potential role of miRNA in temperature-sensitive tospovirus resistance.

Tissue Culture Innovations for Propagation and Conservation of Myrteae-A Globally Important Myrtaceae Tribe.

Myrteae is the most species-rich tribe in the Myrtaceae family, represented by a range of socioeconomically and ecologically significant species. Many of these species, including commercially relevant ones, have become increasingly threatened in the wild, and now require conservation actions. Tissue culture presents an appropriate in vitro tool to facilitate medium-term and long-term wild germplasm conservation, as well as for commercial propagation to maintain desirable traits of commercial cultivars. So far, tissue culture has not been extensively achieved for Myrteae. Here, tissue culture for Eugenia, one of the most species-rich genera in Myrteae, is reviewed, giving directions for other related Myrteae. This review also focuses on ex situ conservation of Australian Myrteae, including using seed banking and field banking. Despite some progress, challenges to conserve these species remain, mostly due to the increasing threats in the wild and limited research. Research into in vitro methods (tissue culture and cryopreservation) is paramount given that at least some of the species are 'non-orthodox'. There is an urgent need to develop long-term in vitro conservation for capturing the remaining germplasm of threatened Myrteae.