tissue culture and virus indexing for the production of clean planting materials

INTRODUCTION  Tissue  culture  is  one  of  the  most  widely  used  biotechnologies  in  African   agricultural   improvement.   When   coupled   with   various  therapies   it  can  be  used  for  elimina;on  of  viruses   from  plants.  The   known   therapies   include:   1.   Thermotherapy   2.  Chemotherapy  and  3.  Electrotherapy.  Thermotherapy  combined  with  meristem  ;p  culture  has  proven  to  be  successful.  However,  the  rate  of  success  can  be  improved  by  combining  two  or  more  different  treatments.  The  Swedish  government,  through  SIDA,  is  suppor;ng   ;ssue   culture   and   diagnos;cs   research   of   the  following   crops:   Taro,   Yam,   Garlic   and   Passion   fruit   from  different  regions  within  eastern  and  central  Africa.  The  aims  of  this  work  are  to  (1)  Iden;fy  the  viruses  present  in  the  plants  (2)  Produce   clean   plan;ng   materials   through   ;ssue   culture   using  one  or  more  of  the  above  men;oned  cleaning  methods,  (3)  pass  clean   plantlets   to   partners   for   mul;plica;on   and   subsequent  distribu;on.  However,  the  above  cleaning  methods  can  be  used  for  other  clonally  propagated  crops.  

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DELIVERING  SCIENCE  AND  DEVELOPING  CAPACITY  Ø   Virus-­‐free  plan;ng  materials  can  be  obtained  using  one  or  a  combina;on  of  the  known  cleaning  therapies.      Ø    Famers   will   have   access   to   disease   -­‐free   plan;ng   materials,   leading   to  increased  crop  yields.  Ø   Diagnos;c  technologies  will  be  transferred  to  na;onal  as  well  as  regional  partners  hence  building  capacity.    

CONCLUSION    

The  results  of  this  study    demonstrate:  Ø   Successful  elimina;on  of  badnavirus  from  taro  using  thermotherapy    combined  with  meristem  ;p  culture.          Ø     Thermotherapy  combined  with  chemotherapy  yielded  beRer  results  hence  the  need  to  combine  cleaning  therapies  for  higher  success  rates.  

Dec  2012  

 METHODOLOGY                                                    

Tissue  culture  and  virus  indexing  for  the  producFon  of  clean  planFng  materials  

P. ASAMI1; C. TOO1; M. MACHARIA1; P. NIYONZIMA2 ; D. BIGIRIMANA3; G. NDARUBAYEMWO3;

D. BEYENE4; J. HARVEY1 and T.A. HOLTON1

1Biosciences eastern and central Africa-International Livestock Research Institute, Nairobi, Kenya (BecA-ILRI); 2Institut des Sciences Agronomiques du Burundi (ISABU); 3University of Burundi, Faculty of Agronomy; 4 Ethiopian Institute of

Agricultural Research (EIAR)

RESULTS  (TARO  CASE  STUDY)  

Samples  collec;on  and  ini;a;on  in  vitro          (before  thermotherapy  treatment)  

Serological  test  (ELISA)      

Molecular  diagnos;cs  (PCR  and  RT-­‐PCR)  

Thermotherapy  treatment  of  all  posi;ves  in  vitro  plantlets  for  20  days  :    §     daily  temperature  :  38°C  §     photoperiod:  16  hours      §     dark  period:  8  hours  at  28°C    §     intensity  con;nued  light:  5000  lux  §     70%  rela;ve  humidity.  

Molecular  diagnos;cs  (PCR  and  RT-­‐PCR)  a^er  thermotherapy  and  meristem  ;p  culture  

Sequencing  and  data  analysis  

Excising    and    culturing  of  meristem  

PCR  for  badna  virus  using  universal    badna  primers.  Badna  amplified:  280bp.  

     M        12      13          14            15          16            17          18            19            W        20            21        22  M              12            13        14                  15              16          17          18            W              19                20              21            22

Badna   PCR   a^er   thermotherapy   and    meristem   ;p   culture   using   universal   badna  primers  

Virus-­‐free  in  vitro  plantlets  

I n   v i t ro   p lant l e t s   a^er   comb ined          thermotherapy  and  meristem  ;p  culture      

Plant  with  viral  symptoms  collected    from  a  taro-­‐growing  field  in  Burundi.  

BEFORE  TREATMENT   AFTER  TREATMENT  

§ Badnavirus  was  detected  in  diseased  plants  prior  to  ;ssue  culture.  

§ 60   in  vitro  plantlets  tested  posi;ve  for  badnavirus:  DNA  sequencing  iden;fied  a  new  suspected  taro  virus  (Dioscorea  bacilliform  virus)  that  has  previously  been  reported  in  yam.    § Thermotherapy   combined   with   chemotherapy   was   able   to   produce   clean  plants,  confirmed  a^er  PCR  and  RT-­‐PCR  diagnos;cs.  

Unlocking livestock development potential through science, influence and capacity development ILRI APM, Addis Ababa, 15-17 May 2013