Plant regeneration from leaf mesophyll-derived protoplasts and gene editing of cassava for enhancement of root shelf-life
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Date
2024-05
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Kenyatta University
Abstract
Manihot esculenta (Crantz) a starchy, multipurpose root crop is grown by millions of smallholder farmers in tropical and subtropical countries. Despite many benefits,
physiological degradation of storage roots which happens within 24–72 hours post-harvest makes them unpleasant and unmarketable, severely constraining its production. The
storage root experiences an oxidative burst and a buildup of secondary metabolites, with scopoletin being the most prevalent, as a result of the inevitable damage that occurs to the
roots during harvest. Blue-black discoloration and vascular streaking observed during deterioration occurs due to over-accumulation of scopoletin whose biosynthesis involves
key enzymes including feruloyl CoA 6′-hydroxylase coded by Manihot esculenta feruloyl CoA 6′-hydroxylase1, 2 and 3 genes. Although plants' genomes have been edited using
tools such as zinc finger and transcription activator-like effector nucleases, these tools are time and labour consuming. Clustered regularly interspaced palindromic repeats
(CRISPR)-Cas technology has provided an effective and successful gene-editing method for crop improvement since it is quicker, less expensive, and better at multiplexing than
other technologies. CRISPR-Cas9 targeted mutagenesis of feruloyl CoA 6′-hydroxylase genes (MeF6'H1, MeF6'H1-2 and MeF6'H3) were used in this study to delay postharvest
physiological deterioration in cassava roots. Three cassava varieties Tms 60444, Muchericheri and Karibuni were used. A reproducible cassava protoplast regeneration
protocol was established. Although cassava protoplast regeneration was not successfully established in a solid culture system, regeneration was successful using a suspension
culture system. Evaluation of optimum cell density for cassava protoplast regeneration showed callus induction in all cell densities employed (1, 2, and 3×105
p/mL). The study established 2×105 p/mL as the best protoplast cell density in the suspension culture system for somatic embryogenesis. To generate cassava plants with increased root shelf-life, CRISPR-Cas9 cassettes with Cas9 gene and sgRNA targeting the three genes were introduced into cassava varieties using Agrobacterium-based gene delivery method and
transgenic plants developed through somatic embryogenesis. The gene constructs altered to incorporate neomycin, hygromycin and basta resistant genes were transformed into
Agrobacterium strain GV3101 facilitating the screening of transgenic events. Polymerase chain reaction (PCR) confirmed integration of transgene in putative transgenic plants while
targeted Sanger sequencing revealed CRISPR-Cas9 induced mutations with substitution and deletions being observed above and below the protospacer adjacent motif of target
genes. The amounts of scopoletin in mutant cassava were evaluated using high performance liquid chromatography (HPLC). Plant targeted for edition of dual genes exhibited significant reduction of scopoletin below HPLC detectable levels and had improved shelf-life compared to one gene knockout events and wildtype plants. Evaluation of phenotypic variations between mutant and wildtype cassava plants revealed mutationevent associated traits including stem and petiole depigmentation, auxiliary budding, clawlike leaf appearance, loss of apical dominance and leaf chlorosis. This study shows a practical and viable method to improve cassava storage root shelf-life. Production of cassava with enhanced shelf-life will stabilize yield production, prevent post-harvest wastage, and improve farmers’ income.
Description
A thesis submitted in fulfillment of the requirements for the award of the degree of doctor of philosophy (biotechnology) in the school of pure and applied sciences of Kenyatta University, May 2024
Supervisors. Prof. Richard Oduor, Prof. Wilton Mbinda and Dr. Cecilia Mweu