A simple and rapid protocol for the genetic transformation of Ensete ventricosum

Jonathan Matheka,Ibsa Merga,Jaindra Nath Tripathi,Endale Gebre,Leena Tripathi

doi:10.1186/s13007-019-0512-y

Jonathan Matheka, Ibsa Merga + Show 3 more

Open Access

https://doi.org/10.1186/s13007-019-0512-y

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Abstract

Enset (Ensete ventricosum), also known as Ethiopian banana, is a food security crop for more than 20 million people in Ethiopia. As conventional breeding of enset is very challenging, genetic engineering is an alternative option to introduce important traits such as enhanced disease resistance and nutritional value. Genetic transformation and subsequent regeneration of transgenic enset has never been reported mainly due to challenges in developing transformation protocols for this tropical species. Agrobacterium-mediated transformation could be a practical tool for the genetic improvement of enset. However, the efficiency of the transformation system depends on several parameters such as plant regeneration, genotype, explant, selection agent and Agrobacterium strains. As a first step towards the development of transgenic enset, a simple and rapid plant regeneration system was developed using multiple buds as explants. Induction and proliferation of multiple buds from shoot tip explants was achieved on Murashige and Skoog (MS) medium supplemented with 5 and 10 mg/l of 6-benzylaminopurine (BAP), respectively. Shoots were regenerated from multiple buds on MS media containing 2 mg/l BAP and 0.2% activated charcoal. Based on the optimized regeneration protocol, an Agrobacterium-mediated transformation method was developed using multiple buds as explants and the binary plasmid pCAMBIA2300-GFP containing the green florescent protein (gfp) reporter gene and neomycin phosphotransferase II (nptII) selection marker gene. Transgenic plantlets were obtained within 4 months at a frequency of about 1.25%. The transgenic lines were validated by PCR analysis using primers specific to the nptII gene. To obtain uniformly transformed plantlets, chimerism was diluted by subculturing and regenerating the transgenic shoots on a selective medium containing kanamycin (150 mg/l) for five cycles. The uniformity of the transgenic plants was confirmed by Southern blot hybridization and RT-PCR analyses on different tissues such as leaf, pseudostem and root of same transgenic plant. In the present study, we report a simple Agrobacterium-mediated transformation system for generating transgenic events of enset. To the best of our knowledge, this is the first report on the stable transformation and regeneration of transgenic events of enset. The transformation system established in this study can be used for the generation of transgenic enset with important traits such as disease resistance.

Highlights

IntroductionEnsete ventricosum (commonly known as enset or the Ethiopian banana) is domesticated and cultivated in Ethiopia
Ensete ventricosum is domesticated and cultivated in Ethiopia
Multiple buds were most efficiently induced on shoot tips in the presence of media with 5 mg/l BAP (BIM2), which resulted in a 1.2 g weight gain per explant after 6 weeks of culture (Fig. 2a)

Summary

Introduction

Ensete ventricosum (commonly known as enset or the Ethiopian banana) is domesticated and cultivated in Ethiopia. It is grown on more than 300,000 ha of land in the central and southwestern parts of the country [1]. About 20 million people depend on it for food [2] It belongs to the family Musaceae within the order Zingiberales and genus Ensete comprising seven species (Ensete homblei, E. perrieri, E. ventricosum, E. livingstonianum, E. glaucum, E. superbum and E. lecongkietii). E. ventricosum can withstand long periods of drought, heavy rains and flooding It can be harvested at any time, even years after the plant reaches maturity, making it an excellent food security crop [4]

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