Robust transformation procedure for the production of transgenic farmer-preferred cassava landraces

<p>Abstract</p> <p>Recent progress in cassava transformation has allowed the robust production of transgenic cassava even under suboptimal plant tissue culture conditions. The transformation protocol has so far been used mostly for the cassava model cultivar 60444 because of its good regeneration capacity of embryogenic tissues. However, for deployment and adoption of transgenic cassava in the field it is important to develop robust transformation methods for farmer- and industry-preferred landraces and cultivars. Because dynamics of multiplication and regeneration of embryogenic tissues differ between cassava genotypes, it was necessary to adapt the efficient cv. 60444 transformation protocol to genotypes that are more recalcitrant to transformation. Here we demonstrate that an improved cassava transformation protocol for cv. 60444 could be successfully modified for production of transgenic farmer-preferred cassava landraces. The modified transformation method reports on procedures for optimization and is likely transferable to other cassava genotypes reportedly recalcitrant to transformation provided production of high quality FEC. Because the three farmer-preferred cassava landraces selected in this study have been identified as resistant or tolerant to cassava mosaic disease (CMD), the adapted protocol will be essential to mobilize improved traits into cassava genotypes suitable for regions where CMD limits production.</p

A method for rapid and homogenous initiation of post-harvest physiological deterioration in cassava storage roots identifies Indonesian cultivars with improved shelf-life performance

Abstract Cassava is the most cultivated and consumed root crop in the world. One of the major constraints to the cassava value chain is the short shelf life of cassava storage roots which is primarily due to the so-called post-harvest physiological deterioration (PPD). The identification of natural sources of PPD tolerance represents a key approach to mitigating PPD losses by generating farmer- and industry-preferred cassava cultivars with prolonged shelf life. In the present study, a PPD assessment method was developed to screen for PPD tolerance in the cassava germplasm. The proposed PPD assessment method displayed a reduced rate of microbial infection and allowed a rapid and homogenous development of typical PPD symptoms in the cassava storage roots. We successfully used the PPD assessment method in combination with an image-based PPD scoring method to identify and characterize PPD tolerance in 28 cassava cultivars from the Indonesian cassava germplasm. Our analysis showed a significant and positive correlation between PPD score and dry matter content (r = 0.589–0.664, p-value < 0.001). Analysis of additional root parameters showed a significant and positive correlation between PPD scores at 2 days post-harvest (dph) and root length (r = 0.388, p-value < 0.05). Our analysis identified at least 4 cultivars displaying a significantly delayed onset of PPD symptoms as compared to the other selected cultivars. The availability of cassava cultivars contrasting for tolerance to PPD will be particularly instrumental to understanding the molecular mechanisms associated with delayed PPD in cassava roots

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

RNAi inhibition of feruloyl CoA 6′-hydroxylase reduces scopoletin biosynthesis and post-harvest physiological deterioration in cassava (Manihot esculenta Crantz) storage roots

Cassava (Manihot esculenta Crantz) is a major world crop, whose storage roots provide food for over 800 million throughout the humid tropics. Despite many advantages as a crop, the development of cassava is seriously constrained by the rapid post-harvest physiological deterioration (PPD) of its roots that occurs within 24–72 h of harvest, rendering the roots unpalatable and unmarketable. PPD limits cassava’s marketing possibilities in countries that are undergoing increased development and urbanisation due to growing distances between farms and consumers. The inevitable wounding of the roots caused by harvesting triggers an oxidative burst that spreads throughout the cassava root, together with the accumulation of secondary metabolites including phenolic compounds, of which the coumarin scopoletin (7-hydroxy-6-methoxy-2H-1-benzopyran-2-one) is the most abundant. Scopoletin oxidation yields a blue-black colour, which suggests its involvement in the discoloration observed during PPD. Feruloyl CoA 6′-hydroxylase is a controlling enzyme in the biosynthesis of scopoletin. The cassava genome contains a seven membered family of feruloyl CoA 6′-hydroxylase genes, four of which are expressed in the storage root and, of these, three were capable of functionally complementing Arabidopsis T-DNA insertion mutants in this gene. A RNA interference construct, designed to a highly conserved region of these genes, was used to transform cassava, where it significantly reduced feruloyl CoA 6′-hydroxylase gene expression, scopoletin accumulation and PPD symptom development. Collectively, our results provide evidence that scopoletin plays a major functional role in the development of PPD symptoms, rather than merely paralleling symptom development in the cassava storage root.ISSN:0167-4412ISSN:1573-502

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 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

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-associated symptoms in cassava storage roots.

<p>A. Storage roots of CBSV-inoculated cv. 60444 cassava at 7 month after stem propagation. B. Storage roots of CBSV-inoculated 60444–Hp 9 cassava line at 7 month after stem propagation.</p