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