Optimisation of a somatic embryogenesis and transformation protocol for farmer-preferred cassava cultivars in Kenya

En Marigi,Wm Thagana,Cm Githunguri ,Ls Karanja ,Dw Miano ,A.e Alakonya ,Mathew Piero Ngugi ,Joel Masanga ,D Kirubi ,M Mwangi ,W M Muiru ,Tl Munga ,R.o Oduor

doi:10.4314/acsj.v24i1.4s

Abstract

Cassava (Manihot esculenta Crantz) is a major food crop in developing countries, and holds potential for industrial use. It is, however, affected by various biotic and abiotic stresses that greatly affect its production. The existing regeneration and transformation protocols are not compatible with all cassava cultivars, thus efficient and robust transformation and regeneration protocols for farmer-preferred cultivars need to be optimised for ease of transfer of novel genes. The objective of this study was to develop an efficient transformation and regeneration protocol for a farmer-preferred Kenyan cassava cultivar. We cultured immature leaf lobe and stem explants on Murashige and Skoog (MS) medium, supplemented with varying concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram and á-naphthalene acetic acid (NAA). Plants were recovered on media with 6- Benzylaminopurine (BAP) and GA3 under a 16 hour light/8 hour darkness photoperiod regime. Results showed high regeneration and transformation frequencies for both cultivars. High frequencies of callus induction (>98%) for both cultivars, were obtained when 2,4-D and Picloram were used. Similarly, both auxins initiated somatic embryogenesis, with Picloram producing the highest frequency of somatic embryos (>92%) in TMS 60444, using stem explants. Gus assays revealed high frequencies of transformation of >77% (TMS 60444) and 60% (Kibanda meno mkubwa). This protocol offers promising perspectives for rapid improvement of these cultivars and, therefore, provides a platform for cleaning planting materials, as well as cassava genetic improvement programmes such as control of viral diseases.Key Words: Cytokinin, Manihot esculenta, regeneration protocol

Highlights

Cassava (Manihot esculenta Crantz), a woody perennial shrub, is a vital food crop for over a billion people, especially in developing countries
Kibanda meno mkubwa, from the Coastal Province of Kenya was selected for the study since it is preferred by farmers because of its high starch levels that enables it fetch high prices in the market
The highest frequency of callus induction using 2,4-D (98.59%) for Kibanda meno mkubwa (KMM) was achieved following culture of stem explants on media supplemented with 4 mg l-1 of the auxin

Summary

Introduction

Cassava (Manihot esculenta Crantz), a woody perennial shrub, is a vital food crop for over a billion people, especially in developing countries It holds potential for production of industrial starch and bioethanol (Chetty et al, 2013). It is estimated that over 77,502 ha of land in Kenya are currently under cassava cultivation (FAO, 2011), yet the crop is threatened by two major diseases: cassava mosaic disease, caused by single stranded DNA geminiviruses; and the cassava brown streak disease, caused by a single stranded RNA ipomivirus (Chetty et al, 2013) Strategies to control these diseases have greatly relied upon conventional improvement programmes, which have encountered massive limitations (Ceballos et al, 2004). This requires optimisation of existing protocols for ease of transfer of novel genes, since existing regeneration and transformation protocols may not be compatible with all the cultivars (Hankoua et al, 2006; Saelim et al, 2006; Elibariki et al, 2014)

Objectives

Methods

Results