Cannabis Synthetic Seeds: An Alternative Approach for Commercial Scale of Clonal Propagation and Germplasm Conservation.

Indoor cannabis (Cannabis sativa) cultivation has been rapidly increasing in many countries after legalization. Besides conventional propagation through cuttings, synthetic seed production provides a competent system for mass propagation, germplasm conservation and international exchange of genetic materials. The present study developed a reliable protocol for cannabis synthetic seed production using encapsulation of nodal segments derived from in vitro or in vivo sources. Synthetic seeds were produced in 3% sodium alginate and 75 mM calcium chloride in Murashige and Skoog (MS) medium and stored under various environmental conditions for up to 150 days. The plantlets regrowth efficiency was monitored on culture media up to 30 days after the storage period. Regrowth rates of 70% and 90% were observed in synthetic seeds from in vitro and in vivo-derived sources, respectively, when stored in 6 °C under 50 µmol s-1 m-2 light for 150 days. Furthermore, addition of acetylsalicylic acid (ASA) to the encapsulation matrix not only postponed precocious germination of synthetic seeds at 22 °C, but also improved the regrowth rate of in vivo-derived synthetic seeds to 100% when they were stored in 6 °C under light. Exposure to light during storage significantly increased shoot length of regrown synseeds when compared to those stored in darkness. This difference in shoot growth disappeared when synseeds were treated with 25 µM ASA. All regenerated plantlets were rooted and acclimatized in sterile rockwool plugs without morphological changes.

Identification of Differential Drought Response Mechanisms in Medicago sativa subsp. sativa and falcata through Comparative Assessments at the Physiological, Biochemical, and Transcriptional Levels.

Alfalfa (Medicago sativa L.) is an extensively grown perennial forage legume, and although it is relatively drought tolerant, it consumes high amounts of water and depends upon irrigation in many regions. Given the progressive decline in water available for irrigation, as well as an escalation in climate change-related droughts, there is a critical need to develop alfalfa cultivars with improved drought resilience. M. sativa subsp. falcata is a close relative of the predominantly cultivated M. sativa subsp. sativa, and certain accessions have been demonstrated to exhibit superior performance under drought. As such, we endeavoured to carry out comparative physiological, biochemical, and transcriptomic evaluations of an as of yet unstudied drought-tolerant M. sativa subsp. falcata accession (PI 641381) and a relatively drought-susceptible M. sativa subsp. sativa cultivar (Beaver) to increase our understanding of the molecular mechanisms behind the enhanced ability of falcata to withstand water deficiency. Our findings indicate that unlike the small number of falcata genotypes assessed previously, falcata PI 641381 may exploit smaller, thicker leaves, as well as an increase in the baseline transcriptional levels of genes encoding particular transcription factors, protective proteins, and enzymes involved in the biosynthesis of stress-related compounds. These findings imply that different falcata accessions/genotypes may employ distinct drought response mechanisms, and the study provides a suite of candidate genes to facilitate the breeding of alfalfa with enhanced drought resilience in the future.

Involvement of the miR156/SPL module in flooding response in Medicago sativa.

The highly conserved plant microRNA, miR156, affects plant development, metabolite composition, and stress response. Our previous research revealed the role of miR156 in abiotic stress response in Medicago sativa exerted by downregulating SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE transcription factors. Here we investigated the involvement and possible mechanism of action of the miR156/SPL module in flooding tolerance in alfalfa. For that, we used miR156 overexpressing, SPL13RNAi, flood-tolerant (AAC-Trueman) and -sensitive (AC-Caribou) alfalfa cultivars exposed to flooding. We also used Arabidopsis ABA insensitive (abi1-2, abi5-8) mutants and transgenic lines with either overexpressed (KIN10-OX1, KIN10-OX2) or silenced (KIN10RNAi-1, KIN10RNAi-2) catalytic subunit of SnRK1 to investigate a possible role of ABA and SnRK1 in regulating miR156 expression under flooding. Physiological analysis, hormone profiling and global transcriptome changes revealed a role for miR156/SPL module in flooding tolerance. We also identified nine novel alfalfa SPLs (SPL1, SPL1a, SPL2a, SPL7, SPL7a, SPL8, SPL13a, SPL14, SPL16) responsive to flooding. Our results also showed a possible ABA-dependent SnRK1 upregulation to enhance miR156 expression, resulting in downregulation of SPL4, SPL7a, SPL8, SPL9, SPL13, and SPL13a. We conclude that these effects induce flooding adaptive responses in alfalfa and modulate stress physiology by affecting the transcriptome, ABA metabolites and secondary metabolism.

Transcriptome-IPMS analysis reveals a tissue-dependent miR156/SPL13 regulatory mechanism in alfalfa drought tolerance.

BACKGROUND: We previously reported on the interplay between miR156/SPL13 and WD40-1/DFR to improve response to drought stress in alfalfa (Medicago sativa L.). Here we aimed to investigate whether the role of miR156/SPL13 module in drought response is tissue-specific, and to identify SPL13-interacting proteins. We analyzed the global transcript profiles of leaf, stem, and root tissues of one-month old RNAi-silenced SPL13 (SPL13RNAi) alfalfa plants exposed to drought stress and conducted protein-protein interaction analysis to identify SPL13 interacting partners. RESULT: Transcript analysis combined with weighted gene co-expression network analysis showed tissue and genotype-specific gene expression patterns. Moreover, pathway analysis of stem-derived differentially expressed genes (DEG) revealed upregulation of genes associated with stress mitigating primary and specialized metabolites, whereas genes associated with photosynthesis light reactions were silenced in SPL13RNAi plants. Leaf-derived DEG were attributed to enhanced light reactions, largely photosystem I, II, and electron transport chains, while roots of SPL13RNAi plants upregulated transcripts associated with metal ion transport, carbohydrate, and primary metabolism. Using immunoprecipitation combined with mass spectrometry (IPMS) we showed that SPL13 interacts with proteins involved in photosynthesis, specialized metabolite biosynthesis, and stress tolerance. CONCLUSIONS: We conclude that the miR156/SPL13 module mitigates drought stress in alfalfa by regulating molecular and physiological processes in a tissue-dependent manner.

The interplay between miR156/SPL13 and DFR/WD40-1 regulate drought tolerance in alfalfa.

BACKGROUND: Developing Medicago sativa L. (alfalfa) cultivars tolerant to drought is critical for the crop's sustainable production. miR156 regulates various plant biological functions by silencing SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. RESULTS: To understand the mechanism of miR156-modulated drought stress tolerance in alfalfa we used genotypes with altered expression levels of miR156, miR156-regulated SPL13, and DIHYDROFLAVONOL-4-REDUCTASE (DFR) regulating WD40-1. Previously we reported the involvement of miR156 in drought tolerance, but the mechanism and downstream genes involved in this process were not fully studied. Here we illustrate the interplay between miR156/SPL13 and WD40-1/DFR to regulate drought stress by coordinating gene expression with metabolite and physiological strategies. Low to moderate levels of miR156 overexpression suppressed SPL13 and increased WD40-1 to fine-tune DFR expression for enhanced anthocyanin biosynthesis. This, in combination with other accumulated stress mitigating metabolites and physiological responses, improved drought tolerance. We also demonstrated that SPL13 binds in vivo to the DFR promoter to regulate its expression. CONCLUSIONS: Taken together, our results reveal that moderate relative miR156 transcript levels are sufficient to enhance drought resilience in alfalfa by silencing SPL13 and increasing WD40-1 expression, whereas higher miR156 overexpression results in drought susceptibility.

COP9 signalosome subunit 5A affects phenylpropanoid metabolism, trichome formation and transcription of key genes of a regulatory tri-protein complex in Arabidopsis.

BACKGROUND: Trichomes and phenylpropanoid-derived phenolics are structural and chemical protection against many adverse conditions. Their production is regulated by a network that includes a TTG1/bHLH/MYB tri-protein complex in Arabidopsis. CSN5a, encoding COP9 signalosome subunit 5a, has also been implicated in trichome and anthocyanin production; however, the regulatory roles of CSN5a in the processes through interaction with the tri-protein complex has yet to be investigated. RESULTS: In this study, a new csn5a mutant, sk372, was recovered based on its altered morphological and chemical phenotypes compared to wild-type control. Mutant characterization was conducted with an emphasis on trichome and phenylpropanoid production and possible involvement of the tri-protein complex using metabolite and gene transcription profiling and scanning electron microscopy. Seed metabolite analysis revealed that defective CSN5a led to an enhanced production of many compounds in addition to anthocyanin, most notably phenylpropanoids and carotenoids as well as a glycoside of zeatin. Consistent changes in carotenoids and anthocyanin were also found in the sk372 leaves. In addition, 370 genes were differentially expressed in 10-day old seedlings of sk372 compared to its wild type control. Real-time transcript quantitative analysis showed that in sk372, GL2 and tri-protein complex gene TT2 was significantly suppressed (p < 0.05) while complex genes EGL3 and GL3 slightly decreased (p > 0.05). Complex genes MYB75, GL1 and flavonoid biosynthetic genes TT3 and TT18 in sk372 were all significantly enhanced. Overexpression of GL3 driven by cauliflower mosaic virus 35S promotor increased the number of single pointed trichomes only, no other phenotypic recovery in sk372. CONCLUSIONS: Our results indicated clearly that COP9 signalosome subunit CSN5a affects trichome production and the metabolism of a wide range of phenylpropanoid and carotenoid compounds. Enhanced anthocyanin accumulation and reduced trichome production were related to the enhanced MYB75 and suppressed GL2 and some other differentially expressed genes associated with the TTG1/bHLH/MYB complexes.

Gene editing by CRISPR/Cas9 in the obligatory outcrossing Medicago sativa.

MAIN CONCLUSION: The CRISPR/Cas9 technique was successfully used to edit the genome of the obligatory outcrossing plant species Medicago sativa L. (alfalfa). RNA-guided genome engineering using Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/Cas9 technology enables a variety of applications in plants. Successful application and validation of the CRISPR technique in a multiplex genome, such as that of M. sativa (alfalfa) will ultimately lead to major advances in the improvement of this crop. We used CRISPR/Cas9 technique to mutate squamosa promoter binding protein like 9 (SPL9) gene in alfalfa. Because of the complex features of the alfalfa genome, we first used droplet digital PCR (ddPCR) for high-throughput screening of large populations of CRISPR-modified plants. Based on the results of genome editing rates obtained from the ddPCR screening, plants with relatively high rates were subjected to further analysis by restriction enzyme digestion/PCR amplification analyses. PCR products encompassing the respective small guided RNA target locus were then sub-cloned and sequenced to verify genome editing. In summary, we successfully applied the CRISPR/Cas9 technique to edit the SPL9 gene in a multiplex genome, providing some insights into opportunities to apply this technology in future alfalfa breeding. The overall efficiency in the polyploid alfalfa genome was lower compared to other less-complex plant genomes. Further refinement of the CRISPR technology system will thus be required for more efficient genome editing in this plant.

MicroRNA156 improves drought stress tolerance in alfalfa (Medicago sativa) by silencing SPL13.

Alfalfa (Medicago sativa) is an important forage crop that is often grown in areas that frequently experience drought and water shortage. MicroRNA156 (miR156) is an emerging tool for improving various traits in plants. We tested the role of miR156d in drought response of alfalfa, and observed a significant improvement in drought tolerance of miR156 overexpression (miR156OE) alfalfa genotypes compared to the wild type control (WT). In addition to higher survival and reduced water loss, miR156OE genotypes also maintained higher stomatal conductance compared to WT during drought stress. Furthermore, we observed an enhanced accumulation of compatible solute (proline) and increased levels of abscisic acid (ABA) and antioxidants in miR156OE genotypes. Similarly, alfalfa plants with reduced expression of miR156-targeted SPL13 showed reduced water loss and enhanced stomatal conductance, chlorophyll content and photosynthetic assimilation. Several genes known to be involved in drought tolerance were differentially expressed in leaf and root of miR156 overexpression plants. Taken together, our findings reveal that miR156 improves drought tolerance in alfalfa at least partially by silencing SPL13.