Strategies to introduce resistance in cassava against viruses causing brown streak disease using sirna
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1 Strategies to introduce resistance in cassava against viruses causing brown streak disease using sirna Beena M Ravindran and Stephan Winter WCRTC Nanning, Guangxi, China, January 2016
2 Most Important diseases on cassava Cassava mosaic disease (CMD) Cassava frogskin disease (CFSD) Cassava brown streak disease (CBSD)
3 Objective To introduce virus resistance in Cassava to study spectrum of resistance durability
4 Approach Identify virus sequences to be targeted Expression of virus derived sequences hp inverted repeat (Waterhouse et al., 1998) Artificial mirna (Schwab et al., 2006) tasirna (de Fellipes et al., 2012) Test all constructs in N. benthamiana Transfer the best constructs to cassava Screen cassava for virus resistance Investigate durability
5 U/ CBSV Genome - diversity CBSV amiiv CBSV all10 ami5 ami Ug 23.7 CBSV mo83.8 ami Ug 23.8 ami Ug 9 CBS_3
6 Inverted repeat construct CsVM CBSV_Mo 83 Ci IV2 CBSV_Mo 83 Ci UCBSV_Ke CP UCBSV_Ke CP poly A
7 ami-rna construct LB nos-terminator kan kmr (npti) nos-promotor pc103 CsVMpromoter CBSV Mo amiiv RIGHT BORDER nos terminator CBSV 83.3 UCBSV Ug23.8 UCBS Ug23.7
8 ta-sirna construct mir173 target site mir173 target site mir173 target site UCBSV/CBSV target sequences nos term mirna173 target site left border poly A spacer nos promoter mirna 173 CsVMpromoter right border left border 2 nptii 3' OCS 35S overdrive pcxx Tassi construct design vector 5809 bp
9 hprna - UCBSV UCBSV_Ke 125 CBSV_Tan70 CBSV_Mo 83
10 ami-rna - CBSV UCBSV_Ke susceptible CBSV_Tan susceptible CBSV_Mo Infection control CBSV_Mo resistant
11 tasi-rna multiple targets - CBSV/UCBSV Transgenic plants CBSV_Mo 83 CBSV_Ug 65 CBSV_Tan 70 UCBSV_Ke 125 Wild-type plants CBSV_Mo 83 CBSV_Ug 65 CBSV_Tan 70 UCBSV_Ke 125
12 Cassava transformation FEC production
13 Cassava transformation Agrobacterium treatment and FEC recovery
14 Cassava transformation Somatic embryogenesis and shoot recovery
15 Cassava transformation Rooting under antibiotic pressure
16 Transgene analyses
17 Infection Test - Grafting
18 Infection Test - Grafting T14p139DSC59_18_Mos 83 T14p139DSC59_08_Ug 65 T14p140DSC59_02_Ke 125 T14p138DSC59_63_Tan 70
19 Summary Observations Introducing U/CBSV sequences into N. benthamiana leads to high level of resistance/ immunity with all strategies used IR, ami-rna, tasi-rna Resistance is highly sequence specific with no difference between the approaches IR nt, ami 21nt, tasi Transgenic cassava with tasi-rna U/CBSV constructs are resistant Outcome Efficient virus screening protocols developed for both cassava and N. benthamiana Transgenic N. benthamiana to verify constructs produced Cassava transformation protocol established
20 Basic Research to Enable Agricultural Development (BREAD) Serological reagents / ELISA Kits Dr. Wulf Menzel Anja Butgereitt Ute Zender Virus collection / Virus reference materisals Dr. Stephan Winter Vera Bicknäse Molecular virology / Virus diagnossis Innovative diagnostic procedures Genome exploration Dr. Wulf Menzel Dr. Stephan Winter Marianne Koerbler Jessica Ponnath Janine Ahrendts Virus/ host relationship Dr. Stephan Winter Dr. Beena M Ravindran Marianne Koerbler Beate Stein Bettina Fürholzner Virus/ vector interaction Dr. Monika Götz Beate Stein
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