Genetic engineering approach in the control of cassava brown streak and bacterial blight diseases
Loading...
Date
2015-12
Authors
Njiru, Joshua Mugendi
Journal Title
Journal ISSN
Volume Title
Publisher
Kenyatta University
Abstract
Cassava is the fifth most important food crop in the world. The importance of
cassava in Africa cannot be overstated. As a drought-tolerant crop that does well in
poor soils, it enhances household food security and is a source of income that
provides livelihood to 800 million people globally. However cassava is faced by
numerous challenges of which pests and diseases are the greatest problems to its
production across East and Central Africa (ECA) sub-regions. Cassava is
vulnerable to at least 20 different viral diseases, cassava brown streak disease
(CBSD), being one of the most damaging viral diseases in East Africa. Cassava
bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam)
on the other hand is the most destructive bacterial disease in all cassava growing
areas of the world. The overall objective of this study was to develop cassava
cultivars that would be resistant to CBSD and CBB through genetic engineering
approaches'. A robust reproducible genetic transformation and regeneration
protocol is a prerequisite for any plant transformation system. Therefore this study
optimized a cultivar independent transformation protocol using friable
embryogenic callus (FEC) as the explant. The optimized protocol was then used to
transform selected cultivars for CBB and CBSD resistance. RNA interference
(RNAi) gene construct p5001 expressing inverted repeats of the full length coat
proteins (FL-CP) from UCBSV and CBSV was used to transform farmer preferred
cultivar TME 14 for CBSD resistance. Hypersensitivity response assisting protein
(Hrap) gene was used to transform cultivar cv. 60444 for CBB resistance. The
transformation frequency for Mkombozi was 20.8%, 32.3% for Albert and 39.6%
for cv. 60444. The developed protocol was repeated inĀ· TME 14 and a
transformation frequency of 47.5% was achieved. Through top graft inoculation,
transgenic TME 14 lines were evaluated for CBSD resistance and 85.7% of the
screened plants were immune to CBSD and did not develop disease symptoms.
After inoculation of Xam in cv. 60444 Hrap transgenic lines, 58.3% of screened
events were totally immune to CBB showing no disease symptoms at 60 days post
inoculation and 33.3% had delayed symptoms with reduced disease severity when
compared to the control non-transgenic plants. Post-transcriptional gene silencing
(PTGS) offer significant potential for controlling RNA plant viruses. In this study,
RNAi approach was used to developing CBSD resistant lines by accumulating
transgenically derived siRNAs in the plants. The constitutive expression of the
Hrap in cv. 60444 generated durable resistance against CBB. These findings show
that CBSD and CBB can be effectively controlled using genetic engineering
approaches' .
Description
A thesis submitted in partial fulfilment of the requirements for the award of the Degree of Doctor of Philosophy (Biotechnology) in the School of Pure and Applied Sciences, Kenyatta University. December 2015