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Kenyatta University (2019)

In Vitro Regeneration and Transformation of Striga Hermonthica (Delile) Benth

Waweru, Duncan Njora

Titre : In Vitro Regeneration and Transformation of Striga Hermonthica (Delile) Benth

Auteur : Waweru, Duncan Njora

Université de soutenance  : Kenyatta University

Grade : Masters of Science (Biotechnology) 2019

Striga is an obligate root hemi-parasitic angiosperm that causes devastating cereal crop losses to millions of smallholder farmers in sub-Saharan Africa (SSA). The parasite causes the greatest effects on infertile soils thereby directly affecting the lives of the poorest subsistence farmers. The parasite attaches to a host crop‘s roots, and exploits its water and nutrients leading to stunted growth and death. Several cultural control methods including ; crop rotation, use of trap crops, intercropping, management of soil fertility and seed based technologies have been practiced for decades with limited success. The molecular and genetic mechanisms underpinning Striga-host interactions are poorly understood mainly because functional genetic tools that require aseptic parasite growth, genetic transformation and subsequent regeneration are complicated by the complex Striga life cycle and the obligate nature of the parasite ; which may have specific host requirements not available in the culture media. This study describes the growth of Striga hermonthica under aseptic conditions. S. hermonthica seeds were surface sterilized in a laminar flow and preconditioned for 14days in the dark at 28ºC. The seeds were then germinated using GR24, a synthetic strigolactone analogue, and then cultured on hormone free Murashige and Skoog (MS) media for 14days. A seedling was formed within 14days which was then sub-cultured to MS media in the light. Striga plants were produced after 28days which proved useful in the provision of clean explants for tissue culture. This study also optimized the conditions for the regeneration of S. hermonthica through somatic embryogenesis and direct organogenesis. The best concentrations of growth regulators for callus induction, with a callus induction frequency of 96.7%, was 2.0 mg/l Naphthaleneacetic acid (NAA) and 0.5 mg/l 6-Benzylaminopurine (BAP). For shoot induction, the optimum concentration was 1 mg/l BAP without NAA, which had a 93.3% shoot induction frequency. With regard to explant type, there were no significant differences in the frequency of callus induction between stem, leaf and whole seedling explants of S. hermonthica. No callus was formed when seed radicals were used. Co-culturing rice, a Striga host, with S. hermonthica calli in the same media did not influence the growth of the parasite. However, the parasite negatively affected the growth of the rice by causing necrotic lesions on the leaves of the rice despite there being no attachment. Finally, Agrobacterium tumefaciens harboring a plasmid with the β glucuronidase (GUS) reporter gene was used to test the transformability of S. hermonthica calli and found that the calli tissues tested positive for GUS staining. The protocols described in this study will enhance further understanding of the complex Striga-host interactions by providing a channel for the functional analysis and validation of Striga genes identified through genomic and transcriptomic analyses.


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