ABSTRACT
Tef is a staple food and a valuable cash crop for millions of people in Ethiopia. Lodging is a major limitation to tef production, and for decades, the development of lodging resistant varieties proved difficult with conventional breeding approaches. We used CRISPR/Cas9 to introduce knockout mutations in the tef orthologue of the rice SEMIDWARF-1 (SD-1) gene to confer semidwarfism and ultimately lodging resistance. High frequency recovery of transgenic and SD-1 edited tef lines was achieved in two tef cultivars by Agrobacterium-mediated delivery into young leaf explants of gene editing reagents along with transformation and regeneration enhancing morphogenic genes, BABY BOOM (BBM) and WUSCHEL2 (WUS2). All of the 23 lines analyzed by next-generation sequencing had at least two or more alleles of SD-1 mutated. Of these, 83% had tetra-allelic frameshift mutations in the SD-1 gene in primary tef regenerants, which were inherited in subsequent generations. Phenotypic data generated on T1 and T2 generations revealed that the sd-1 lines have reduced culm and internode lengths with no reduction in either panicle or peduncle lengths. These characteristics are comparable with rice sd-1 plants. Measurements of lodging, in greenhouse-grown plants, showed that sd-1 lines have significantly higher resistance to lodging at the heading stage compared with the controls. This is the first demonstration of the feasibility of high frequency genetic transformation and CRISPR/Cas9-mediated genome editing in this highly valuable but neglected crop. The findings reported here highlight the potential of genome editing for the improvement of lodging resistance and other important traits in tef.
Subject(s)
Eragrostis , Genes, Plant , Alleles , CRISPR-Cas Systems , Eragrostis/genetics , Gene Editing , Mutation , Plant Breeding , Plants, Genetically Modified/geneticsABSTRACT
Enset (Ensete ventricosum (Welw.) Cheesman) is a drought tolerant, vegetatively propagated crop that was domesticated in Ethiopia. It is a staple food for more than 20 million people in Ethiopia. Despite its current importance and immense potential, enset is among the most genetically understudied and underexploited food crops. We collected 230 enset wild and cultivated accessions across the main enset producing regions in Ethiopia and applied amplified fragment length polymorphism (AFLP) and genotype by sequencing (GBS) analyses to these accessions. Wild and cultivated accessions were clearly separated from each other, with 89 genes found to harbour SNPs that separated wild from cultivated accessions. Among these, 17 genes are thought to be involved in flower initiation and seed development. Among cultivated accessions, differentiation was mostly associated with geographical location and with proximity to wild populations. Our results indicate that vegetative propagation of elite clones has favoured capacity for vegetative growth at the expense of capacity for sexual reproduction. This is consistent with previous reports that cultivated enset tends to produce non-viable seeds and flowers less frequently than wild enset.
ABSTRACT
Enset (Ensete ventricosum), also known as Ethiopian banana, is a food security crop for more than 20 million people in Ethiopia. As conventional breeding of enset is very challenging, genetic engineering is an alternative option to introduce important traits such as enhanced disease resistance and nutritional value. Genetic transformation and subsequent regeneration of transgenic enset has never been reported mainly due to challenges in developing transformation protocols for this tropical species. Agrobacterium-mediated transformation could be a practical tool for the genetic improvement of enset. However, the efficiency of the transformation system depends on several parameters such as plant regeneration, genotype, explant, selection agent and Agrobacterium strains. As a first step towards the development of transgenic enset, a simple and rapid plant regeneration system was developed using multiple buds as explants. Induction and proliferation of multiple buds from shoot tip explants was achieved on Murashige and Skoog (MS) medium supplemented with 5 and 10 mg/l of 6-benzylaminopurine (BAP), respectively. Shoots were regenerated from multiple buds on MS media containing 2 mg/l BAP and 0.2% activated charcoal. Based on the optimized regeneration protocol, an Agrobacterium-mediated transformation method was developed using multiple buds as explants and the binary plasmid pCAMBIA2300-GFP containing the green florescent protein (gfp) reporter gene and neomycin phosphotransferase II (nptII) selection marker gene. Transgenic plantlets were obtained within 4 months at a frequency of about 1.25%. The transgenic lines were validated by PCR analysis using primers specific to the nptII gene. To obtain uniformly transformed plantlets, chimerism was diluted by subculturing and regenerating the transgenic shoots on a selective medium containing kanamycin (150 mg/l) for five cycles. The uniformity of the transgenic plants was confirmed by Southern blot hybridization and RT-PCR analyses on different tissues such as leaf, pseudostem and root of same transgenic plant. In the present study, we report a simple Agrobacterium-mediated transformation system for generating transgenic events of enset. To the best of our knowledge, this is the first report on the stable transformation and regeneration of transgenic events of enset. The transformation system established in this study can be used for the generation of transgenic enset with important traits such as disease resistance.
ABSTRACT
Enset (Ensete ventricosum (Welw.) Cheesman) is one of the Ethiopia's indigenous sustainability crops supporting the livelihoods of about 20 million people, mainly in the densely populated South and Southwestern parts of the country. Enset serves as a food security crop for humans, animal feed, and source of fiber for the producers. The production of enset has been constrained by plant pests, diseases, and abiotic factors. Among these constraints, bacterial wilt disease has been the most important limiting factor for enset production since its outbreak five decades ago. There is no known bacterial wilt disease resistant genetic material in the enset genetic pool to transfer this trait to susceptible enset varieties through conventional breeding. Moreover, the absence of effective chemicals against the disease has left farmers without means to combat bacterial wilt for decades. Genetic engineering has been the alternative approach to develop disease resistant plant materials in other crops where traditional breeding tools are ineffective. This review discusses enset cultivation and recent developments addressing the control of bacterial wilt disease in enset and related crops like banana to help design effective strategies.
ABSTRACT
Enset (Ensete ventricosum (Welw.) Cheesman) is an economically important staple food crop in Ethiopia, especially in the southern and southwestern regions. It is called "false banana" due to its resemblance to banana, but inability to produce any edible fruit. The crop is clonally propagated using field-grown suckers. This study reports the development of a robust regeneration technique to propagate large numbers of plantlets using corm discs containing intercalary meristematic tissues. Hundreds of shoot buds were induced from corm discs of enset cultivar 'Bedadeti' cultured on Murashige and Skoog (MS) medium supplemented with 1.5 mg L-1 2,4-dichlorophenoxyacetic acid, 0.216 mg L-1 zeatin, and 2 g L-1 activated charcoal. The shoot buds were regenerated into complete plantlets when transferred onto MS medium supplemented with 1 mg L-1 6-benzylaminopurine and 2 g L-1 activated charcoal. More than 100 plantlets were generated in 4 mo from corm discs isolated from a single in vitro mother plantlet. Well-rooted plantlets were acclimatized in soil with 100% success, and did not show any apparent phenotypic abnormalities under glasshouse conditions. This efficient regeneration system could be very useful for the rapid multiplication of clean pathogen-free planting material.
