Characterization of brown streak virus-resistant cassava

Cassava brown streak disease (CBSD) has become a major constraint to cassava production in East and Central Africa. The identification of new sources of CBSD resistance is essential to deploy CBSD mitigation strategies as the disease is progressing westwards to new geographical areas. A stringent infection method based on top cleft grafting combined with precise virus titer quantitation was utilized to screen fourteen cassava cultivars and elite breeding lines. When inoculated with mixed infections of Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), the scions of elite breeding lines KBH 2006/18 and KBH 2006/26 remained symptom-free during a 16-week period of virus graft inoculation, while susceptible varieties displayed typical CBSD infection symptoms at 4 weeks after grafting. The identified CBSD resistance was stable under the co-inoculation of CBSV, UCBSV with cassava geminiviruses (CGMs). Double grafting experiments revealed that transmission of CBSV and UCBSV to CBSD susceptible top scions was delayed when using intermediate scions of elite breeding lines KBH 2006/18 and KBH 2006/26. Nonetheless, comparison of virus systemic movement using scions from KBH2006/18 and a transgenic CBSD resistant 60444 line (60444-Hp9 line) showed that both CBSV and UCBSV move at undetectable levels through the stems. Further, protoplast-based assays of virus titers over time showed that the replication of CBSVs is inhibited in the resistant line KBH2006/18, suggesting that the identified CBSD resistance is at least partially based on inhibition of virus replication. Our molecular characterization of CBSD resistance in cassava offers a robust virus–host system to further investigate the molecular determinants of CBSD resistance

Current progress and challenges in crop genetic transformation

Plant transformation remains the most sought-after technology for functional genomics and crop genetic improvement, especially for introducing specific new traits and to modify or recombine already existing traits. Along with many other agricultural technologies, the global production of genetically engineered crops has steadily grown since they were first introduced 25 years ago. Since the first transfer of DNA into plant cells using Agrobacterium tumefaciens, different transformation methods have enabled rapid advances in molecular breeding approaches to bring crop varieties with novel traits to the market that would be difficult or not possible to achieve with conventional breeding methods. Today, transformation to produce genetically engineered crops is the fastest and most widely adopted technology in agriculture. The rapidly increasing number of sequenced plant genomes and information from functional genomics data to understand gene function, together with novel gene cloning and tissue culture methods, is further accelerating crop improvement and trait development. These advances are welcome and needed to make crops more resilient to climate change and to secure their yield for feeding the increasing human population. Despite the success, transformation remains a bottleneck because many plant species and crop genotypes are recalcitrant to established tissue culture and regeneration conditions, or they show poor transformability. Improvements are possible using morphogenetic transcriptional regulators, but their broader applicability remains to be tested. Advances in genome editing techniques and direct, nontissue culture-based transformation methods offer alternative approaches to enhance varietal development in other recalcitrant crops. Here, we review recent developments in plant transformation and regeneration, and discuss opportunities for new breeding technologies in agriculture.ISSN:0176-1617ISSN:1618-132

The influence of Prandtl number on near-wall turbulent heat transfer

Za opis turbulentnega prenosa toplote iz stene na tekočino je pri nizkih vrednostih Reynoldsovih in Prandtlovih številih mogoče uporabiti neposredno numerično simulacijo (NNS-DNS), ki opiše vse krajevne in časovne skale pojava.Vpliv Reynoldsovega števila na turbulentni prenos toplote (hitrosti, temperature, fluktuacije itn.) je razmeroma majhen, medtem ko je vpliv Prandtlovega števila veliko večji, in sicer Pr=0.025, Pr=1 in Pr=5.4. Ločljivost NNS turbulentnega prenosa gibalne količine je premo sorazmerna z Re 3/4 v vseh smereh koordinatnega sistema. Pri upoštevanju prenosa toplote, za tekočine s Prandtlovim številom, večjim od ena, velja, da je ločljivost premo sorazmerna z Re 3/4Pr 1/2. Pri Re=5260 in Pr=5.4 smo opravili tri numerične simulacije pri različnih ločljivostih. Vse tri simulacije so NNS za hitrostno polje , samo simulacija z največjo ločljivostjo je tudi NNS za temperaturno polje. Rezultati so pokazali, da je mogoče temperaturno polje zelo natančno napovedati tudi z nekoliko slabšo ločljivostjo od teoretično zahtevane.For describing the heat transfer from a wall to a fluid at low Reynolds and Prandtl numbers we can use a direct numerical simulations (DNS), which describes all the length and time scales of the phenomenon. The Reynolds number has a weak influence on the turbulent heat transfer (velocities, temperatures, RMS-fluctuations ...), where as the increasing Prandtl number has a stronger influence. In our flow simulations in the channel, three different Prandtl numbers, i.e. Pr=0.025, Pr=1 and Pr=5.4, et a Reynolds number=5000 were analyzed. The resolution of the DNS for turbulent momentum transfer is proportional to Re 3/4 in all directions. When considering heat transfer in fluids for a Prandtl number higher than one, the resolution is proportional to Re 3/4Pr 1/2. Three diferent numerical simulations at different resolutions were performed at Re=5260 and Pr=5.4. All three simulations are a DNS for the velocity field,whereas only the simulation at the highest resolution is also a DNS for the thermal field. The results showed that the thermal field could be accurately described with a lower resolution than theoretically required

CBSV and UCBSV-associated symptoms in cassava leaves.

<p>A. Fully expanded leaf of wild-type TME 7 scion grafted on a CBSV-infected rootstock at 10 wpg. B. Fully expanded leaf of wild-type TME 7 scion grafted on an UCBSV-infected rootstock at 10 wpg. C. Fully expanded leaf of TME 7–Hp 9 scion grafted on a CBSV-infected rootstock at 10 wpg. D. Fully expanded leaf of TME 7 scion grafted on an UCBSV-infected rootstock at 10 wpg.</p

CBSV and UCBSV quantitation in rootstocks and TME 7 scions.

<p>A. CBSV quantitation by qPCR in CBSV-infected cv. 60444 roostock plants and corresponding grafted scions from transgenic, wild-type and control TME 7 lines. B. UCBSV quantitation by qPCR in UCBSV-infected Ebwanateraka roostock plants and corresponding grafted scions from transgenic, wild-type and control TME 7 lines. Numbers following the cassava line identifiers indicate the biological replicates.</p

CBSV and UCBSV quantitation in rootstocks and cv. 60444 scions.

<p>A. CBSV quantitation by qPCR in CBSV-infected cv. 60444 roostock plants and corresponding grafted scions from transgenic, wild-type and control cv. 60444 lines. B. UCBSV quantitation by qPCR in UCBSV-infected Ebwanateraka roostock plants and corresponding grafted scions from transgenic, wild-type and control cv. 60444 lines. Numbers following the cassava line identifiers indicate the biological replicates.</p

Phenotypic and molecular data of the 60444-Hp scion-propagated plants.

<p>Stem cuttings from inoculated scions were propagated. Leaves and roots were evaluated 3 and 7 months after propagation, respectively.</p

Linking CRISPR-Cas9 interference in cassava to the evolution of editing-resistant geminiviruses

BACKGROUND: Geminiviruses cause damaging diseases in several important crop species. However, limited progress has been made in developing crop varieties resistant to these highly diverse DNA viruses. Recently, the bacterial CRISPR/Cas9 system has been transferred to plants to target and confer immunity to geminiviruses. In this study, we use CRISPR-Cas9 interference in the staple food crop cassava with the aim of engineering resistance to African cassava mosaic virus, a member of a widespread and important family (Geminiviridae) of plant-pathogenic DNA viruses. RESULTS: Our results show that the CRISPR system fails to confer effective resistance to the virus during glasshouse inoculations. Further, we find that between 33 and 48% of edited virus genomes evolve a conserved single-nucleotide mutation that confers resistance to CRISPR-Cas9 cleavage. We also find that in the model plant Nicotiana benthamiana the replication of the novel, mutant virus is dependent on the presence of the wild-type virus. CONCLUSIONS: Our study highlights the risks associated with CRISPR-Cas9 virus immunity in eukaryotes given that the mutagenic nature of the system generates viral escapes in a short time period. Our in-depth analysis of virus populations also represents a template for future studies analyzing virus escape from anti-viral CRISPR transgenics. This is especially important for informing regulation of such actively mutagenic applications of CRISPR-Cas9 technology in agriculture.status: publishe