De novo transcriptomic analysis of Doum Palm (Hyphaene compressa) revealed an insight into its potential drought tolerance.

BACKGROUND: Doum palms (Hyphaene compressa) perform a crucial starring role in the lives of Kenya's arid and semi-arid people for empowerment and sustenance. Despite the crop's potential for economic gain, there is a lack of genetic resources and detailed information about its domestication at the molecular level. Given the doum palm's vast potential as a widely distributed plant in semi-arid and arid climates and a source of many applications, coupled with the current changing climate scenario, it is essential to understand the molecular processes that provide drought resistance to this plant. RESULTS: Assembly of the first transcriptome of doum palms subjected to water stress generated about 39.97 Gb of RNA-Seq data. The assembled transcriptome revealed 193,167 unigenes with an average length of 1655 bp, with 128,708 (66.63%) successfully annotated in seven public databases. Unigenes exhibited significant differentially expressed genes (DEGs) in well-watered and stressed-treated plants, with 45071 and 42457 accounting for up-regulated and down-regulated DEGs, respectively. GO term, KEGG, and KOG analysis showed that DEGs were functionally enriched cellular processes, metabolic processes, cellular and catalytic activity, metabolism, genetic information processing, signal transduction mechanisms, and posttranslational modification pathways. Transcription factors (TF), such as the MYB, WRKY, NAC family, FAR1, B3, bHLH, and bZIP, were the prominent TF families identified as doum palm DEGs encoding drought stress tolerance. CONCLUSIONS: This study provides a complete understanding of DEGs involved in drought stress at the transcriptome level in doum palms. This research is, therefore, the foundation for the characterization of potential genes, leading to a clear understanding of its drought stress responses and providing resources for improved genetic modification.

Plant regeneration from leaf mesophyll derived protoplasts of cassava (Manihot esculenta Crantz).

A high yield of isolated protoplast and reliable regeneration system are prerequisite for successful somatic hybridization and genome editing research. However, reproducible plant regeneration from protoplasts remains a bottleneck for many crops, including cassava. We evaluated several factors that influence isolation of viable protoplasts form leaf mesophyll, induction of embryogenic calli, and regeneration of plants in three cassava cultivars; Muchericheri, TMS60444 and Karibuni. A relatively higher protoplast yield was obtained with enzyme mixture containing 5 g/L Macerozyme and 10 g/L cellulase. Muchericheri recorded relatively higher protoplast yield of 20.50±0.50×106 whereas TMS60444 (10.25±0.25×106) had the least protoplast yield in 10 g/L cellulase and 4 g/L cellulase. Freshly isolated protoplast cells were plated on callus induction medium (CIM) solid medium containing MS basal salt, 60 g/L D-glucose, 30 g/L sucrose, B5 vitamins, 100 mg/L myo-inositol, 0.5 mg/L copper sulphate, 100 mg/L casein hydrolysate, 4.55 g/L mannitol, 0.1 g/L MES, 10 mg/L picloram and 3 g/L gelrite to induce protoplast growth and development. The three cultivars reached colony formation but no further development was observed in this culture method. Protoplast growth and development was further evaluated in suspension culture using varying cell densities (1, 2 and 3× 105 p/mL). Development with highest number of minicalli was observed in cell density of 3× 105 p/mL. Minicalli obtained were cultured on CIM supplemented with 10mg/L picloram. Callus induction was observed in all cell densities with the cultivars. Highest somatic embryogenesis was observed in 2× 105 p/ml while no somatic embryogenesis was observed in cell density of 1×105 p/mL. Somatic embryos were matured in EMM medium supplemented with 1 mg/L BAP, 0.02 mg/L NAA and 1.5 mg/L GA3 then germinated in hormone free medium for plant regeneration. This protocol which used simple mixture of commercial enzymes is highly reproducible and can be applied in biotechnology research on cassava.

Targeted mutagenesis of the CYP79D1 gene via CRISPR/Cas9-mediated genome editing results in lower levels of cyanide in cassava.

Cassava is the world's most essential food root crop, generating calories to millions of Sub-Saharan African subsistence farmers. Cassava leaves and roots contain toxic quantities of the cyanogenic glycoside linamarin. Consumption of residual cyanogens results in cyanide poisoning due to conversion of the cyanogens to cyanide in the body. There is a need for acyanogenic cassava cultivars in order for it to become a consistently safe and acceptable food, and commercial crop. In recent years, the CRISPR/Cas system, has proven to be the most effective and successful genome editing tool for gene function studies and crop improvement. In this study, we performed targeted mutagenesis of the MeCYP79D1 gene in exon 3, using CRISPR/Cas9, via Agrobacterium-mediated transformation. The vector design resulted in knockout in cotyledon-stage somatic embryos regenerated under hygromycin selection. Eight plants were recovered and genotyped. DNA sequencing analysis revealed that the tested putative transgenic plants carried mutations within the MeCYP79D1 locus, with deletions and substitutions being reported upstream and downstream of the PAM sequence, respectively. The levels of linamarin and evolved cyanide present in the leaves of mecyp79d1 lines were reduced up to seven-fold. Nevertheless, the cassava linamarin and cyanide were not completely eliminated by the MeCYP79D1 knockout. Our results indicate that CRISPR/Cas9-mediated mutagenesis is as an alternative approach for development of cassava plants with lowered cyanide content.

Genetic Diversity and Population Structure of Doum Palm (Hyphaene compressa) Using Genotyping by Sequencing.

Doum palm (Hyphaene compressa) is a perennial economic plant primarily growing in Kenya's Arid and Semi-Arid Lands (ASALs). It is heavily relied upon for food, animal feed, construction materials and medicine, making it an ideal plant for resource sustainability. However, the limited information on its genetic resources has hindered its breeding and conservation studies. This study used the genotyping by sequencing approach to identify Single Nucleotide Polymorphisms. These SNPs were further used to assess the genetic diversity and population structure of 96 H. compressa accessions from Coastal, Northern and Eastern ASAL regions of Kenya using two approaches; reference-based and de novo-based assemblies. STRUCTURE analysis grouped the sampled accessions into two genetic clusters (Cluster 1 and Cluster 2). Cluster 1 included accessions from the Northern region, whereas Cluster 2 included all accessions from Eastern and Coastal regions. Accessions from Kwale (Coastal) had mixed ancestry from both Cluster 1 and Cluster 2. These STRUCTURE findings were further supported by principal components analysis, discriminant analysis of principal components and phylogenetic analysis. Analysis of molecular variance indicated greater genetic variation within populations (92.7%) than among populations (7.3%). An overall FST of 0.074 was observed, signifying moderate genetic differentiation among populations. The results of this study will provide information useful in breeding, marker-assisted selection and conservation management of H. compressa.

Phenotypic Diversity of Doum Palm (Hyphaene compressa), a Semi-Domesticated Palm in the Arid and Semi-Arid Regions of Kenya.

Hyphaene compressa is an economically important palm in Africa. Despite its significant role in the livelihoods of rural communities, the diversity of doum palm is poorly documented and studied. In addition, it has no model descriptor that can aid such studies. Ninety H. compressa accessions collected from Northern, Eastern, and Coastal regions of Kenya were examined to determine the morphological variability of the vegetative and fruit traits of H. compressa and to identify its morphotypes for improvement. A total of 19 morphological characters including seven quantitative and 12 qualitative traits of fruit and vegetative traits were selected. Linear mixed-effects models, principal component analysis, and linear discriminant analyses were used to assess the variation in the morphological traits of doum palm based on the regions. Hierarchical clustering was performed to identify the morphotypes of H. compressa. There was variability in H. compressa morphological traits, particularly at the Kenyan Coast. All seven quantitative traits were able to effectively discriminate doum palm phenotypically (p ≤ 0.001). The 90 accessions clustered into five morphotypes designated as 1, 2, 3, 4, and 5. Morphotype 4 was specific only to the Coastal region. Morphotype 5 had the tallest trees with the biggest fruits and included palms from Eastern and Coastal regions making it the best morphotype for fruit traits. This study will inform the domestication, improvement, and conservation of H. compressa by selecting elite accessions.

Differential characterization of physiological and biochemical responses during drought stress in finger millet varieties.

Drought is the most perilous abiotic stress that affects finger millet growth and productivity worldwide. For the successful production of finger millet, selection of drought tolerant varieties is necessary and critical stages under drought stress, germination and early seedling growth, ought to be fully understood. This study investigated the physiological and biochemical responses of six finger millet varieties (GBK043137, GBK043128, GBK043124, GBK043122, GBK043094 and GBK043050) under mannitol-induced drought stress. Seeds were germinated in sterile soil and irrigated with various concentrations of mannitol (200, 400 and 600 mM) for 2 weeks. In a comparative analysis relative water content (RWC), chlorophyll, proline and malondialdehyde (MDA) contents were measured to obtain the physiological and biochemical characteristics of drought stress. The results showed that increased levels of drought stress seriously decreased germination and early seedling growth of finger millet varieties. However, root growth was increased. In addition, exposition to drought stress triggered a significant decrease in relative water content and chlorophyll content reduction, and the biochemical parameters assay showed less reduction in RWC. Furthermore, oxidative damage indicating parameters, such as proline concentration and MDA content, increased. Varieties GBK043137 and GBK043094 were less affected by drought than the other varieties as shown by significant changes in their physiological parameters. Our findings reveal the differences between the physiological and biochemical responses of finger millet to drought and are vital for breeding and selecting drought tolerant varieties of finger millet. Further, genomic and molecular investigations need to be undertaken to gain a deeper insight into the detailed mechanisms of drought tolerance in finger millet.