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.

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.

Finger millet is one of the most important cereals that are often grown in semiarid and arid regions of East-Africa. Salinity is known to be a major impediment for the crop growth and production. This study was aimed to understand the mechanisms of physiological and biochemical responses to salinity stress of Kenyan finger millet varieties (GBK043137, GBK043128, GBK043124, GBK043122, GBK043094, GBK043050) grown across different agroecological zones under NaCl-induced salinity stress. Seeds were germinated on the sterile soil and treated using various concentrations of NaCl (100, 200 and 300 mM) for two weeks. Again, the earlyseedling stage of germinated plants was irrigated with the same salt concentrations for 60 days. Results indicated depression in germination percentage, shoot and root growth rate, leaf relative water content, chlorophyll content contents, leaf K + concentration, and leaf K + /Na + ratios increased salt levels. Contrary, proline and malonaldehyde (MDA) contents reduced sugar content and leaf total proteins. At the same time, the leaf Na + and Clamounts of all plants increased substantially with rising stress levels. Clustering analysis revealed that GBK043094 and GBK043137 were placed together and identified as salt-tolerant varieties based on their performance under salt stress. Overall, our findings indicated a significant varietal variability for most of the parameters analysed. These superior varieties identified could be potentially used as promising genetic resources in future breeding programmes development directed towards salt-tolerant finger millet hybrids. Further analysis at genomic level need to be undertaken to better understand the genetic factors that promote salinity tolerance in finger millet.

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 on sterile soil and irrigated with various concentrations of mannitol (200, 400 and 600 mM) for two weeks. Comparative analysis in terms of relative water content (RWC), chlorophyll, proline, and malondialdehyde (MDA) contents were measured the physiological and biochemical characteristics of drought stress. The results showed that increased level 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 the biochemical parameters assay showed less reduction of relative water content. Furthermore, oxidative damage indicating parameters such as proline concentration and MDA content increased. Varieties GBK043137 and GBK043094 were less affected by drought as shown by significant change in the physiological parameters. Our findings reveal the difference and linkage between the physiological responses of finger millet to drought and are vital for breeding and selection of drought tolerant varieties of finger millet. Further investigations on genomic and molecular to deeply insight the detail mechanisms of drought tolerance in finger millet need to explored.

Salinity stress is a major environmental impediment affecting the growth and production of crops. Finger millet is an important cereal grown in many arid and semi-arid areas of the world characterized by erratic rainfall and scarcity of good-quality water. Finger millet salinity stress is caused by the accumulation of soluble salts due to irrigation without a proper drainage system, coupled with the underlying rocks having a high salt content, which leads to the salinization of arable land. This problem is projected to be exacerbated by climate change. The use of new and efficient strategies that provide stable salinity tolerance across a wide range of environments can guarantee sustainable production of finger millet in the future. In this review, we analyze the strategies that have been used for salinity stress management in finger millet production and discuss potential future directions toward the development of salt-tolerant finger millet varieties. This review also describes how advanced biotechnological tools are being used to develop salt-tolerant plants. The biotechnological techniques discussed in this review are simple to implement, have design flexibility, low cost, and highly efficient. This information provides insights into enhancing finger millet salinity tolerance and improving production.

Cassava is a major food crop for millions of people in Africa, Asia and South America, forming an essential food-security and income generation commodity for small-scale or subsistence farming communities. The storage root is the most important component of the crop that provides more calories than cereals. Immediately after harvest, cassava storage roots undergo complex biochemical and physiological changes known as postharvest physiological deterioration (PPD), which is influenced by genotype, environmental and agronomic factors, resulting to spoilage, rendering the storage roots unpalatable and unmarketable. This problem has remained unresolved over the years. This review describes the innovative breeding technologies which could be used to prolong cassava storage root shelf-life. In this review, we discuss the available knowledge on (i) physiology and biochemistry of cassava storage root with regard to PPD (ii) strategies for minimizing PPD in cassava storage roots (iii) traits associated with PPD tolerance as essential targets for prolonging cassava storage root shelf life, and (iv) suggestions for novel genomic tools and modern genetic and breeding approaches for prolonging shelf-life in cassava storage roots. With its extensive genomic resources including the public release of cassava reference genome sequence assembly and other and resources, and innovative plant breeding technologies, the crop offers an excellent opportunity to serve as a model to address postharvest spoilage and improve food security. Continuous improvements based on the new plant breeding technologies (genome editing, speeding breeding and RNA-dependent DNA methylation) in cassava and innovations in postharvest handling and storage of the storage roots are expected to provide sustainable solutions for PPD constraints and make cassava an important food security and nutrition and industrial crop.

A simple and efficient plant regeneration system via direct organogenesis was established in finger millet using in vitro derived shoot apical meristems. Six varieties; GBK-043128, GBK-043094, GBK-043050, GBK-043137, GBK-043122 and GBK-043124 were evaluated. MS medium was used for cotyledonary germination. Maximum number of shoots (84.33%) was observed in variety GBK-043128 while GBK-043094 had the least germination efficiency (62. 67%). Shoot apical meristems from threeday old seedlings were evaluated for their potency of shoot induction on varied 6-benzylaminopurine (BAP) concentrations. Highest shoot induction was observed in medium supplemented with 1.75 mg/l BAP in GBK-043050 (3.00) whereas GBK-043094 (1.28) had the least response in medium supplemented with 1.0 mg/l BAP. To induce rooting, in vitro regenerated plants cultured on MS medium was supplemented with different concentrations of indole-3-acetic acid. The highest response in root induction, with a larger number of roots (10.28), was observed in MS medium supplemented with 4.0 µM IAA. Statistical analysis indicated that plant regeneration response varied greatly among the varieties. In vitro germinated plants were successfully transferred to the greenhouse after hardening, with 300 shoots developing into fertile plants, which were indistinguishable with wild type plants. This plant regeneration system has potential for production of transgenic finger millet crops.

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.