Optimization of protoplast isolation,transformation and its application in sugarcane (Saccharum spontaneum L.)

Sugarcane is a prominent source of sugar and ethanol production.Genetic analysis for trait improvement of sugarcane is greatly hindered by its complex genome,long breeding cycle,and recalcitrance to genetic transformation.The protoplast-based transient transformation system is a versatile and convenient tool for in vivo functional gene analysis;however,quick and effective transformation systems are still lacking for sugarcane.Here,we developed an efficient protoplast-based transformation system by optimizing conditions of protoplasts isolation and PEG-mediated transformation in S.spontaneum.The yield of viable protoplasts was approximately 1.26×107 per gram of leaf material,and the transformation efficiency of 80.19%could be achieved under the optimized condition.Furthermore,using this approach,the nuclear localization of an ABI5-like bZIPs transcription factor was validated,and the promoter activity of several putative DNase I hypersensitive sites(DHSs)was assessed.The results indicated this system can be conveniently applied to protein subcellular localization and promoter activity assays.A highly efficient S.spontaneum mesophyll cell protoplast isolation and transient transformation method was developed,and it shall be suitable for in vivo functional gene analysis in sugarcane.

First report of field resistance to cyantraniliprole, a new anthranilic diamide insecticide, on Bemisia tabaciMED in China

The Bemisia tabaci(Gennadius)cryptic species complex comprises important insect pests that cause devastating damage to agricultural crops worldwide.In China,the B.tabaci Mediterranean(MED)(or biotype Q)species is threatening agricultural production all over the country as resistance to commonly used insecticides has increased.This situation highlights the need for alternative pest control measures.Cyantraniliprole,a novel anthranilic diamide insecticide,has been widely employed to control Hemipteran pests.To monitor the levels of resistance to cyantraniliprole in B.tabaci field populations in China,bioassays were conducted for 18 field samples from nine provinces over two years.Compared with median lethal concentration(LC_(50))for the MED susceptible strain,all field samples had significantly higher resistance to cyantraniliprole.Furthermore,resistance factors(RFs)increased significantly in samples from Shanxi(from 5.62 in 2015 to 25.81 in 2016),Hunan(3.30 in 2015 to 20.97 in 2016)and Hubei(from 9.81 in 2015 to 23.91 in 2016)provinces.This study indicates a considerable decrease in the efficacy of cyantraniliprole against B.tabaci and establishes a baseline of susceptibility that could serve as a reference for future monitoring and management of B.tabaci resistance to cyantraniliprole.

Genetics and fitness costs of resistance to flupyradifurone in Bemisia tabaci from China

Flupyradifurone is a promising new insecticide used for controlling Bemisia tabaci during vegetable production.In this study,we assessed the fitness costs and mode of inheritance associated with resistance to flupyradifurone in B.tabaci by comparing the susceptible strain(MED-S)to one field-evolved flupyradifurone-resistant strain(WH-R,with 199-fold resistance)and one laboratory-selected flupyradifurone-resistant strain(FLU-SEL,with 124-fold resistance).Progenies of reciprocal crosses between WH-R and MED-S(F_(1A),F_(1B),and pooled F_(1)),and between FLU-SEL and MED-S(F_(1C),F_(1D),and pooled F_(1)’),showed varying degrees of dominance,indicating that resistance to flupyradifurone in WH-R was autosomal and incompletely dominant,yet in FLU-SEL it was autosomal and incompletely recessive.Furthermore,the development of resistance to flupyradifurone occurred at the expense of fitness costs for the resistant populations.Compared to the MED-S strain,WH-R showed a relative fitness of 0.50 with significantly prolonged developmental durations and reduced survival rates of the nymphal and pseudopupal stages,as well as decreased fecundity and hatchability.Similarly,FLU-SEL showed a relative fitness of 0.65 and also demonstrated prolonged developmental durations and reduced survival rates of nymphs and pseudopupae,as well as decreased hatchability in comparison with the MED-S strain.However,no significant differences in fecundity were observed between MED-S and FLU-SEL.The present study provides useful knowledge for formulating pest management strategies in the field,which will allow growers to slow the development of resistance to flupyradifurone and to sustainably control B.tabaci.