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.

A Review of Recent Advances and Future Directions in the Management of Salinity Stress in Finger Millet.

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.

Varietal differences in physiological and biochemical responses to salinity stress in six finger millet plants.

Finger millet is an important cereal that is grown in semi-arid and arid regions of East-Africa. Salinity stress is a major environmental impediment for the crop growth and production. This study aimed to understand the physiological and biochemical responses to salinity stress of six Kenyan finger millet varieties (GBK043137, GBK043128, GBK043124, GBK043122, GBK043094, GBK043050) grown across different agroecological zones under NaCl-induced salinity stress (100, 200 and 300 mM NaCl). Seeds were germinated on the sterile soil and treated using various concentrations of NaCl for 2 weeks. Early-seedling stage of germinated plants were irrigated with the same salt concentrations for 60 days. The results indicated depression in germination percentage, shoot and root growth rate, leaf relative water content, chlorophyll content, leaf K+ concentration, and leaf K+/Na+ ratios with increased salt levels and the degree of increment differed among the varieties. On the contrary, the content of proline, malonaldehyde, leaf total proteins, and reduced sugar increased with increasing salinity. At the same time, the leaf Na+ and Cl- amounts of all plants increased substantially with increasing stress levels. Clustering analysis placed GBK043094 and GBK043137 together and these varieties were identified as salt-tolerant based on their performance. Taken together, our findings indicated a significant varietal variability for most of the parameters analysed. The superior varieties identified could be used as promising genetic resources in future breeding programmes directed towards development of salt-tolerant finger millet hybrids. Further analysis at genomic level needs to be undertaken to better understand the genetic factors that promote salinity tolerance in finger millet.

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.