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

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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

 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

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

Were, HK1, Ememwa I1, Wabwile MW1, Were MN1,Vernderschuren H2 and Gruissem W2

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 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

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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

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Symptoms of CBSD and CMD

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).

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 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

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 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)

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 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

WHAT WE DID . . . . . . . . . . . .2

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Transformed cassava in glass house In tissue culture In the CFT In the lab

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Transgenic cassava growing at Alupe CFT

Line Description of event Mean % CMD incidence Mean tuber count (No.) Mean Tuber weight (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

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 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

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

• pen

n circul ular ar

30 12 9 141 145 133 167 166 137 30 30 12 9 129 141 141 145 145 133 16 7 167 166 166 137 137 348 348 Ladder

SOU OUTHERN HERN BLOT OT WITH H AN ENDOG OGENO ENOUS US PROB OBE (LOA OADING DING CON ONTROL) OL)

3021.25 1401.878 1853.11 4697.736 664.2388 167.466 1494.076 12 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 60444 129 133 167 348 166 137 q

CMV TITRE qPCR

PP2A CMV(all) F-GGTCCTGGATTGCAGAGGAAGATAGTGGG R-GGTACAAACGTCATTGATGACGTCGATCCC

qPCR RESULTS

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

CBSV TITRE (qPCR)

30 141 145 133 167 166 137 30 30 12 9 129 141 141 145 145 133 16 7 167 166 166 137 137 +

• 91% of the samples tested were EACMV positive

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

 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

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 Financial support was provided by ETH-Zurich  Work done in collaboration with Prof. Wilhelm

Gruissem, ETH-Zurich

 NBA

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