6 th Annual National Biosafety Conference, KSMS 3 rd - 6 th October, 2017

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1 Evaluation of Transformed cassava Lines for Resistance to CBSD and CMD in Kenya 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, 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

2 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

3 Were HK PhD 3

4 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

5 Symptoms of CBSD and CMD Were HK PhD 5

6 diseased healthy

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8 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?

9 Line TME 7-has natural resistance against CMD Line 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

10 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

11 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 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

12 WHAT WE DID 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

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

14 Transgenic cassava growing at Alupe CFT Were HK PhD 14

15 Line Description of event Mean % CMD incidence Mean tuber count (No.) Mean Tuber weight (kg) 74 ds CR ds ACI ds ACI ds AVI double single ds ACI ds AVI Pc k ds ACI ds ACI ds ACI ds AVI ds ACI ds AVI double single

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

17 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

18 LADDER ds open circular ds close circular ssdna

19 SOUTHERN BLOT WITH AN ENDOGENOUS PROBE (LOADING CONTROL) 30 Ladder

20 qpcr RESULTS CMV TITRE qpcr q PP2A CMV(all) F-GGTCCTGGATTGCAGAGGAAGATAGTGGG R-GGTACAAACGTCATTGATGACGTCGATCCC

21 9 8 CBSV TITRE (qpcr) PSTV NEGV

22 % of the samples tested were EACMV positive

23 Ladder - 75%-ACMV positive 2/3 sample negative in lines 129,166 and 167

24 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

25 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

26 Were HK PhD 26

27 Were HK PhD 27

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

29 Were HK PhD 29