CRISPR/Cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding

Open Access Journal; Published online: 31 Jan 2019Presence of the integrated endogenous banana streak virus (eBSV) in the B genome of plantain (AAB) is a major challenge for breeding and dissemination of hybrids. As the eBSV activates into infectious viral particles under stress, the progenitor Musa balbisiana and its derivants, having at least one B genome, cannot be used as parents for crop improvement. Here, we report a strategy to inactivate the eBSV by editing the virus sequences. The regenerated genome-edited events of Gonja Manjaya showed mutations in the targeted sites with the potential to prevent proper transcription or/and translational into functional viral proteins. Seventy-five percent of the edited events remained asymptomatic in comparison to the non-edited control plants under water stress conditions, confirming inactivation of eBSV into infectious viral particles. This study paves the way for the improvement of B genome germplasm and its use in breeding programs to produce hybrids that can be globally disseminated

Effective charging energy for a regular granular metal array

We study the Ambegaokar-Eckern-Sch\"{o}n (AES) model for a regular array of metallic grains coupled by tunnel junctions of conductance $g$ and calculate both paramagnetic and diamagnetic terms in the Kubo formula for the conductivity. We find analytically, and confirm by numerical path integral Monte Carlo methods, that for $0<g<4$ the conductivity obeys an Arrhenius law $\sigma(T)\sim\exp[-E^{*}(g)/T]$ with an effective charging energy $E^{*} (g)$ when the temperature is sufficiently low, due to a subtle cancellation between $T^2$ inelastic-cotunneling contributions in the paramagnetic and diamagnetic terms. We present numerical results for the effective charging energy and compare the results with recent theoretical analyses. We discuss the different ways in which the experimentally observed $\sigma(T)\sim\exp[-\sqrt{T_{0}/T}]$ law could be attributed to disorder.Comment: 5 pages, 3 figures, ReVTeX; added estimates of effective charging energies and discussion of effects of disorde

Relative susceptibility of banana cultivars to Xanthomonas campestris pv. musacearum

The banana Xanthomonas wilt (BXW) is the most devastating disease of banana in Great Lakes region of East and Central Africa. The pathogen kills plants quickly and spreads rapidly over a large area in ashort time making the disease one of the most dreaded in banana. The disease affects almost all varieties of commonly grown banana cultivars. Some knowledge of the relative susceptibility of banana cultivars would be extremely useful and could be a basis for management strategies for BXW. Ten banana cultivars were evaluated for their relative susceptibility to Xanthomonas campestris pv. musacearum. All the ten cultivars were tested by injecting bacterial inoculum in pseudostem of in vitro plantlets as well as potted plants. The various banana cultivars showed significant differences in susceptibility to Xanthomonas campestris pv. musacearum. The beer banana cultivar ‘Pisang Awak’ and dessert cultivars ‘Dwarf Cavendish’, ‘Giant Cavendish’, and ‘FHIA-17’ were found to be highlysusceptible, where as East African Highland banana cultivar ‘Nakitembe’ was found to be least susceptible. The other cultivars ‘Mpologoma’, ‘Mbwazirume’, ‘Sukali Ndiizi’ and ‘FHIA-25’, were found to be susceptible. Diploid parent ‘Musa balbisiana’ was found to be resistant. This study clearly highlights the need for development of new resistant cultivars for BXW disease control, as all the commercial cultivars are susceptible

Plant regeneration from axillary bud derived callus in white yam (Dioscorea rotundata)

Published online: 14 June 2016Dioscorea rotundata, commonly known as white yam, is an important staple food crop widely cultivated in West Africa and provides food security to millions of people. Genetic improvements of this crop using the advanced biotechnology tools have been hampered hitherto by the recalcitrant nature of regeneration through somatic embryogenesis. Therefore, we have developed an efficient and reproducible system for plant regeneration via somatic embryogenesis. Explants of different types (immature leaf, node, stem internode, root segment, petiole, and axillary bud) of D. rotundata accession TDr 2436 were tested for their embryogenic potentials on Murashige and Skoog (MS) medium supplemented with various auxins (2,4-D, NAA, and picloram). Among all explants tested, axillary bud explants cultured on MS medium supplemented with picloram (0.5–12 mg/l) favored the production of calli. Maximum proliferation of calli (526 mg fresh weight/explant) was achieved on MS medium supplemented with picloram (0.5 mg/l), casein hydrolysate (600 mg/l), and proline (1 g/l). Histology analysis confirmed that the embryogenic calli produced on this medium were mixed with non-embryogenic calli. Transfer of calli on MS basal medium supplemented with activated charcoal (1 %) changed the color of calli to purple and promoted the production of somatic embryos (87 embryos/callus) as well as adventitious shoot buds. Furthermore, upon transfer to MS medium supplemented with BAP (0.4 mg/l), the embryos continued their differentiation and maturation and germinated into complete plantlets. The adventitious shoot buds produced multiple shoots on MS medium supplemented with BAP (0.4 mg/l). Well-developed germinated plantlets were acclimatized in the screen house with 90 % survivability. Histology studies confirmed that the regeneration of D. rotundata reported here followed dual regeneration pathways. The embryogenic calli regenerated through development of somatic embryos and germinated into complete plantlets, however non-embryogenic calli regenerated through organogenesis and developed multiple shoots. The developed protocol has potential for somatic hybridization, mass clonal propagation, and genetic transformation applications

Transgenics in crop improvement research at IITA

Biotechnology has opened unprecedented avenues for exploring biological systems. Transgenics is one of the key techniques particularly useful for the genetic improvement of crops that are not amenable to conventional breeding, such as those that are vegetatively propagated. In IITA, transgenic technologies are being used for improving banana/plantain (Musa sp.), cassava (Manihot esculenta), and yam (Dioscorea sp.)

A high-throughput regeneration and transformation platform for production of genetically modified banana

Published online: 2015 Nov 26Banana (Musa spp.) is an important staple food as well as cash crop in tropical and subtropical countries. Various bacterial, fungal, and viral diseases and pests such as nematodes are major constraints in its production and are currently destabilizing the banana production in sub-Saharan Africa. Genetic engineering is a complementary option used for incorporating useful traits in banana to bypass the long generation time, polyploidy, and sterility of most of the cultivated varieties. A robust transformation protocol for farmer preferred varieties is crucial for banana genomics and improvement. A robust and reproducible system for genetic transformation of banana using embryogenic cell suspensions (ECS) has been developed in this study. Two different types of explants (immature male flowers and multiple buds) were tested for their ability to develop ECS in several varieties of banana locally grown in Africa. ECS of banana varieties “Cavendish Williams” and “Gros Michel” were developed using multiple buds, whereas ECS of “Sukali Ndiizi” was developed using immature male flowers. Regeneration efficiency of ECS was about 20,000–50,000 plantlets per ml of settled cell volume (SCV) depending on variety. ECS of three different varieties were transformed through Agrobacterium-mediated transformation using gusA reporter gene and 20–70 independent transgenic events per ml SCV of ECS were regenerated on selective medium. The presence and integration of gusA gene in transgenic plants was confirmed by PCR, dot blot, and Southern blot analysis and expression by histochemical GUS assays. The robust transformation platform was successfully used to generate hundreds of transgenic lines with disease resistance. Such a platform will facilitate the transfer of technologies to national agricultural research systems (NARS) in Africa

Agrobacterium-mediated transformation of plantain (Musa spp.) cultivar Agbagba

An Agrobacterium-mediated plant transformation system was developed for the production of transgenic plantain [Musa spp. cultivar Agbagba (AAB)]. Apical shoot tips were transformed using Agrobacterium strain EHA105 with the binary vector pCAMBIA 1201, having the hygromycin resistancegene as a selection marker and GUS-INT as a reporter gene. Transient expression of the bglucuronidase (uid A) gene was achieved in transformed apical shoot tips. The hygromycin resistant shoots were regenerated 4 to 5 weeks after co-cultivation of explants with Agrobacterium. The two stepselection procedure allowed the regeneration of shoots which were uniformly transformed. The integration of the uid A gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. In this study, transformation based on regeneration from apical shoot tips has beendemonstrated. This process does not incorporate steps using disorganized cell cultures but uses micropropagation, which has the important advantage that it allows regeneration of homogeneous populations of plants in a short period of time. This study shows the enormous potential for geneticmanipulation of Musa species for disease and pest resistance, as well as abiotic factors, using a rapid and non-species specific transformation and regeneration system

Techniques for detecting genetically modified crops and products

The cultivation of genetically modified crops is becoming increasingly important; more traits are emerging and more acres than ever before are being planted with GM varieties. The release of GM crops and products in the markets worldwide has increased the regulatory need to monitor and verify the presence and the amount of GM varieties in crops and products. Labeling legislation and trade requirements differ from one country to another, leading to the necessity for the development of reliable and sensitive analytical methods for detection, identification and quantification of GM varieties in crops and their products. GM crops and their products can be identified by detecting either the inserted genetic material at DNA level, the resulting protein or phenotype. Several analytical methods such as methods based on the polymerase chain reaction (PCR) for detecting the inserted DNA, immunological assays for detecting the resulting protein, or using bioassays to detect the resultant phenotype have been developed. So far only PCR has found broad application in GMO detection as a generally accepted method for regulatory purposes. Presently, real-time PCR can be considered as the most powerful tool for the detection and quantification of GM crops and products

Agrobacteriummediated genetic transformation of yam (Dioscorea rotundata): an important tool for functional study of genes and crop improvement

Published online: 15 Sep 2014Although genetic transformation of clonally propagated crops has been widely studied as a tool for crop improvement and as a vital part of the development of functional genomics resources, there has been no report of any existing Agrobacterium-mediated transformation of yam (Dioscorea spp.) with evidence of stable integration of T-DNA. Yam is an important crop in the tropics and subtropics providing food security and income to over 300 million people. However, yam production remains constrained by increasing levels of field and storage pests and diseases. A major constraint to the development of biotechnological approaches for yam improvement has been the lack of an efficient and robust transformation and regeneration system. In this study, we developed an Agrobacterium-mediated transformation of Dioscorea rotundata using axillary buds as explants. Two cultivars of D. rotundata were transformed using Agrobacterium tumefaciens harboring the binary vectors containing selectable marker and reporter genes. After selection with appropriate concentrations of antibiotic, shoots were developed on shoot induction and elongation medium. The elongated antibiotic-resistant shoots were subsequently rooted on medium supplemented with selection agent. Successful transformation was confirmed by polymerase chain reaction, Southern blot analysis, and reporter genes assay. Expression of gusA gene in transgenic plants was also verified by reverse transcription polymerase chain reaction analysis. Transformation efficiency varied from 9.4 to 18.2% depending on the cultivars, selectable marker genes, and the Agrobacterium strain used for transformation. It took 3–4 months from Agro-infection to regeneration of complete transgenic plant. Here we report an efficient, fast and reproducible protocol for Agrobacterium-mediated transformation of D. rotundata using axillary buds as explants, which provides a useful platform for future genetic engineering studies in this economically important crop