CMD in in Ke Kenya ya Were, HK 1 , Ememwa I 1 , Wabwile MW 1 , - PowerPoint PPT Presentation

Eva valua uation tion of f Tra ransform sformed ed cas assav ava a Li Lines es fo for r Resistance stance to CBSD SD an and CMD in in Ke Kenya ya Were, HK 1 , Ememwa I 1 , Wabwile MW 1 , Were MN 1 ,Vernderschuren H 2 and Gruissem W 2  1 Department of Biological Sciences, Masinde Muliro University of Science and Technology, P.O. Box 190, 50100 Kakamega, Kenya.  2 Department of Biology and Plant Biotechnology, ETH Zurich, Universitaetstrasse 2, 8092 Zurich Switzerland 6 th Annual National Biosafety Conference, KSMS 3 rd - 6 th October, 2017 Were HK PhD 1

 Cassava (Mannihot esculenta Crantz) is starchy, tuberous root crop  Is grown mainly in western Kenya (including Kakamega, Bungoma, Busia, Homa Bay, Siaya, Kisii, Migori Makueni, Kilifi and Kwale counties  It is usually grown in rain fed, low-input systems and the yields obtained in Kenya of 4 t/ha are among the poorest in the world  Although traditionally regarded as a subsistence crop, cassava is increasingly being produced for commercial purposes Were HK PhD 2

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 Pests and diseases are major problems: ◦ Cassava Mosaic Disease (CMD) ◦ Cassava brown streak disease (CBSV) ◦ Cassava Bacterial blight ( Xanthomonas axonopodis PV manihotis ) ◦ Cassava anthracnose ( Colletotrichum gloeosporoides f.sp manihotis )  Inadequate or Lack of high quality disease-free planting material in the form of ‘seed’ cuttings  Low soil fertility and poor agronomic practices Were HK PhD 4

Symptoms of CBSD and CMD Were HK PhD 5

diseased healthy

 Two viral diseases: (ACMV, a DNA virus) and (CBSV, an RNA virus), often resulting in total loss of starchy root yield  Breeding for resistance is difficult, time-consuming and often not durable  So far no natural resistance to CBSV  Genetic engineering may stand a chance?

 Line TME 7-has natural resistance against CMD  Line 60444-has some tolerance to CBSD  Virus resistance can be achieved by expression of inverted repeat sequences coding for hairpin double-stranded RNAs homologous to viral sequences.  The hairpin RNAs target viral sequences for degradation and/or modifications.  Such small RNAs homologous to viruses do accumulate in wild-type cassava under natural infection (Akbergenov et al., 2006). Were HK PhD 9

 Develop cassava that is resistant to both CMD and CBSD using replicase and RNAi technology (genetic engineering)  Evaluate the engineered clones in the glass house and in the Field for resistance to the viruses Were HK PhD 10

 Friable embryogenic callus (FEC) generated according to (Bull et al., 2009; Zainuddin et al., 2012)  FEC genetically transformed with Agrobacterium tumefasciens LBA4404 carrying the binary vectors for transformation.  Line TME 7-engineered for resistance to CBSD  Line 60444-engineered for resistance to CMD  Transgenic cassava lines regenerated from transformed FEC following established procedures (Zhang et al., 2000; Bull et al., 2009; Zainuddin et al., 2012) Were HK PhD 11

WHAT WE DID . . . . . . . . . . . .2  Regenerated transformed cassava plantlets hardened in the glass house for 60 days  Scions from transgenic cassava grafted on diseased cassava rootstocks in the greenhouse  Through greenhouse inoculation assays, the transgenic cultivars remained resistant in successive planting cycles Were HK PhD 12

In tissue culture Transformed cassava in glass house In the lab In the CFT Were HK PhD 13

Transgenic cassava growing at Alupe CFT Were HK PhD 14

Line Description of event Mean % Mean Tuber CMD Mean tuber weight incidence count (No.) (kg) 74 ds CR-2 93 2 0.1 133 ds ACI-152 76 3 0.3 157 ds ACI ds AVI-113 94 9 0.5 167 double single 53 59 8 0.8 145 ds ACI ds AVI-59 20 27 0.7 348 Pc 1301 -2k 85 5 0.6 30 60444 82 6 0.4 115 ds ACI-2 75 7 0.5 129 ds ACI-101 54 5 0.9 137 ds ACI ds AVI-7 76 5 0.5 141 ds ACI ds AVI-55 79 5 0.5 166 double single 52 71 7 0.9

• Six lines (19, 402, 407, 501, 506 and 30) had both foliage and root symptoms • Nine lines (22, 56, 398.401,404, 406,497 and 498, 499) had only root symptoms • Lines 405 and 506 had neither root nor foliar symptoms 16

 Two lines ( 405 and 506) show promising response towards CBSD resistance  Inserting a construct in TME line breaks CMD resistance  Not yet clear if we have CMD resistance in the plants Were HK PhD 17

ER ADDER 348 167 129 141 145 LAD 348 141 167 129 145 ds o open n circul ular ar ds c close circul ular ssDNA NA

OBE US PROB 348 ENOUS 348 137 OGENO 137 137 OL) ONTROL) 166 H AN ENDOG 166 166 16 DING CON 167 7 167 133 133 OADING OT WITH 145 145 145 141 (LOA 141 HERN BLOT 141 129 12 OUTHERN 12 9 30 9 30 30 SOU Ladder

qPCR RESULTS CMV TITRE qPCR 5000 4500 4697.736 4000 3500 3000 3021.25 2500 2000 1853.11 1500 1494.076 1401.878 1000 500 664.2388 167.466 12 0 60444 129 133 167 348 166 137 q PP2A CMV(all) F-GGTCCTGGATTGCAGAGGAAGATAGTGGG R-GGTACAAACGTCATTGATGACGTCGATCCC

CBSV TITRE (qPCR) 9 8 8.1667445 1667445 7 6 5 4 3 2 1.98542 98542966 9667 1.4913673 491367333 1 1.347841 347841 1.32005 32005 0.9108545 9108545 0.798935 798935 0.2134818 2134818 0.357682 357682 0.025519 025519 0 6044 60444 401 401 501 501 506 506 402 402 407 407 499 499 406 406 PS PSTV TV NEGV

+ 91% of the samples tested were EACMV positive - 137 137 137 166 166 166 16 167 7 167 133 133 145 145 145 141 141 141 129 12 9 30 30 30

- + 137 137 2/3 sample negative in lines 129,166 and 167 137 166 166 166 16 167 7 167 133 133 133 145 145 145 141 141 75%-ACMV positive 141 129 12 12 9 30 9 30 30 Ladder

 Mixed ACMV-EACMV infection in 62% of the samples tested  72% of the ACMV positive samples tested positive for ACMV-Ug Severe  61% positive for ACMV-Ug 34  86% of the EACMV positive samples were positive for EACMV-UG  None of the samples was positive EACMV-TZ and EACMV-Ke  UCBSV was more prevalent (82%) than CBSV (67%)  Most samples had mixed infections of CBSV and UCBSV  The viruses EACMV-TZ, EACMCV, EACMNV, EACMKV and EACMZV were not detected

 For CMD, Lines 129 and 166 show some tolerance  Most lines infected by more than one virus species (high virus titres)  High disease and vector pressure in W Kenya  For CBSD, lines 405 and 506 showed some resistance and need to be investigated further  Both CBSD and UCBSV occur in Western Kenya 25

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 Financial support was provided by ETH-Zurich  Work done in collaboration with Prof. Wilhelm Gruissem, ETH-Zurich  NBA Were HK PhD 28

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