Resistance to Sri Lankan Cassava MosaicVirus (SLCMV) in Genetically Engineered Cassava cv. KU50 through RNA Silencing, by Valentine Otang Ntui, Kynet Kong, Raham Sher Khan, Tomoko Igawa, Gnanaguru Janaky Janavi , Ramalingam Rabindran, Ikuo Nakamura , Masahiro Mii, PLOS ONE | DOI:10.1371/journal.pone.0120551 April 22, 2015
Description:
Abstract
Cassava ranks fifth among the starch producing crops of the world, its annual bioethanol yield is higher than for any other crop. Cassava cultivar KU50, the most widely grown culti- var for non-food purposes is susceptible to Sri Lankan cassava mosaic virus (SLCMV). The objective of this work was to engineer resistance to SLCMV by RNA interference (RNAi) in order to increase biomass yield, an important aspect for bioethanol production. Here, we produced transgenic KU50 lines expressing dsRNA homologous to the region between the AV2 and AV1 of DNA A of SLCMV. High level expression of dsRNA of SLCMV did not in- duce any growth abnormality in the transgenic plants. Transgenic lines displayed high lev- els of resistance to SLCMV compared to the wild-type plants and no virus load could be detected in uninoculated new leaves of the infected resistant lines after PCR amplification and RT-PCR analysis. The agronomic performance of the transgenic lines was unimpaired after inoculation with the virus as the plants presented similar growth when compared to the mock inoculated control plants and revealed no apparent reduction in the amount and weight of tubers produced. We show that the resistance is correlated with post-transcription- al gene silencing because of the production of transgene specific siRNA. The results dem- onstrate that transgenic lines exhibited high levels of resistance to SLCMV. This resistance coupled with the desirable yield components in the transgenic lines makes them better can- didates for exploitation in the production of biomass as well as bioethanol.
Cassava ranks fifth among the starch producing crops of the world, its annual bioethanol yield is higher than for any other crop. Cassava cultivar KU50, the most widely grown culti- var for non-food purposes is susceptible to Sri Lankan cassava mosaic virus (SLCMV). The objective of this work was to engineer resistance to SLCMV by RNA interference (RNAi) in order to increase biomass yield, an important aspect for bioethanol production. Here, we produced transgenic KU50 lines expressing dsRNA homologous to the region between the AV2 and AV1 of DNA A of SLCMV. High level expression of dsRNA of SLCMV did not in- duce any growth abnormality in the transgenic plants. Transgenic lines displayed high lev- els of resistance to SLCMV compared to the wild-type plants and no virus load could be detected in uninoculated new leaves of the infected resistant lines after PCR amplification and RT-PCR analysis. The agronomic performance of the transgenic lines was unimpaired after inoculation with the virus as the plants presented similar growth when compared to the mock inoculated control plants and revealed no apparent reduction in the amount and weight of tubers produced. We show that the resistance is correlated with post-transcription- al gene silencing because of the production of transgene specific siRNA. The results dem- onstrate that transgenic lines exhibited high levels of resistance to SLCMV. This resistance coupled with the desirable yield components in the transgenic lines makes them better can- didates for exploitation in the production of biomass as well as bioethanol.
Transgenic Tobacco Lines Expressing Defective CMV Replicase-Derived dsRNA Are Resistant to CMV-O and CMV-Y, by Valentine Otang Ntui, Kong Kynet, Raham Sher Khan, Mari Ohara, Yasuko Goto, Manabu Watanabe, Masanobu Fukami, Ikuo Nakamura, Masahiro Mii. Mol Biotechnol DOI 10.1007/s12033-013-9681-5
Description:
Abstract
Cucumber mosaic virus (CMV) is a tripartite, positive sense RNA virus causing infections and yield losses to many plant species. Here, we generated a construct containing inverted repeat of 1,793 bp fragment of defective CMV replicase gene derived from RNA2 of cucumber mosaic virus strain O (CMV-O). The replicase gene was modified by deleting a 9 bp region between nucleotides 1909–1918. This caused a deletion in the active centre motif of polymerases, producing defective translated product 9 nucleotides shorter than the full length protein. The RNAi construct containing inverted repeat of the defective gene was used to produce transgenic tobacco lines expressing CMV-derived double-stranded RNA via Agrobacterium-mediated transformation. Of the four transgenic lines inoculated with CMV-O or CMV-Y in vitro and ex vivo, three lines (T1, T4 and T5) showed immunity to both strains of CMV as no symptoms were detected, whereas one line (T7) exhibited high resistance with mild symptoms limited to inoculation portions. No virus could be detected in uninoculated new leaves of the transgenic lines after RT-PCR and Dot-immunobinding assay analyses. Small interfering RNAs present in transgenic lines before and after virus challenge indicates that the resistance was acquired through RNA silencing.
Cucumber mosaic virus (CMV) is a tripartite, positive sense RNA virus causing infections and yield losses to many plant species. Here, we generated a construct containing inverted repeat of 1,793 bp fragment of defective CMV replicase gene derived from RNA2 of cucumber mosaic virus strain O (CMV-O). The replicase gene was modified by deleting a 9 bp region between nucleotides 1909–1918. This caused a deletion in the active centre motif of polymerases, producing defective translated product 9 nucleotides shorter than the full length protein. The RNAi construct containing inverted repeat of the defective gene was used to produce transgenic tobacco lines expressing CMV-derived double-stranded RNA via Agrobacterium-mediated transformation. Of the four transgenic lines inoculated with CMV-O or CMV-Y in vitro and ex vivo, three lines (T1, T4 and T5) showed immunity to both strains of CMV as no symptoms were detected, whereas one line (T7) exhibited high resistance with mild symptoms limited to inoculation portions. No virus could be detected in uninoculated new leaves of the transgenic lines after RT-PCR and Dot-immunobinding assay analyses. Small interfering RNAs present in transgenic lines before and after virus challenge indicates that the resistance was acquired through RNA silencing.
Nutrient balances for cassava cultivation in Kampong Cham province in Northeast Cambodia, by Sopheap, U Patanothai, A Aye, Tin Maung , U. Sopheap et al. / International Journal of Plant Production (2012) 6(1): 37-58
Description:
Abstract
In Cambodia, cassava is mostly grown with little or no fertilizer inputs, but the magnitudes of nutrient balances are not known. This study was conducted to assess nutrient balances for cassava cultivation in Kampong Cham province in Northeast Cambodia. Forty-five households in four cassava production zones were interviewed in relation to their cultural practices and crop residue management, upon which sources of nutrient inputs and outputs were based. Chemical fertilizer, manure, planting materials and rainfall were the defined inputs, while cassava roots and stumps were the outputs. Crop cutting was undertaken in the cassava fields of the 45 households to obtain weights of roots and other plant parts. Nutrient balances were calculated for the individual fields based on nutrient contents of the component sources obtained from the literatures. The results showed negative balances for all the nutrients evaluated. The imbalances were most serious for N, K and Ca with the averages of -64.45 kg N, -52.83 kg K and -10.83 kg Ca ha-1, but were less serious for P and Mg with the averages of -2.85 kg P and -7.20 kg Mg ha-1. These negative balances were the consequence of low nutrient inputs in current practices where only a few farmers applied low rates of chemical fertilizer or manure. Continued use of current practices will threaten the sustainability of cassava production in Cambodia. For long term productivity of the crop, the application of organic manures together with lime and chemical fertilizers high in N and K is recommended.
In Cambodia, cassava is mostly grown with little or no fertilizer inputs, but the magnitudes of nutrient balances are not known. This study was conducted to assess nutrient balances for cassava cultivation in Kampong Cham province in Northeast Cambodia. Forty-five households in four cassava production zones were interviewed in relation to their cultural practices and crop residue management, upon which sources of nutrient inputs and outputs were based. Chemical fertilizer, manure, planting materials and rainfall were the defined inputs, while cassava roots and stumps were the outputs. Crop cutting was undertaken in the cassava fields of the 45 households to obtain weights of roots and other plant parts. Nutrient balances were calculated for the individual fields based on nutrient contents of the component sources obtained from the literatures. The results showed negative balances for all the nutrients evaluated. The imbalances were most serious for N, K and Ca with the averages of -64.45 kg N, -52.83 kg K and -10.83 kg Ca ha-1, but were less serious for P and Mg with the averages of -2.85 kg P and -7.20 kg Mg ha-1. These negative balances were the consequence of low nutrient inputs in current practices where only a few farmers applied low rates of chemical fertilizer or manure. Continued use of current practices will threaten the sustainability of cassava production in Cambodia. For long term productivity of the crop, the application of organic manures together with lime and chemical fertilizers high in N and K is recommended.
