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

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.

MaMAPK3-MaICE1-MaPOD P7 pathway, a positive regulator of cold tolerance in banana

“…Purified DNA was digested with EcoR V and fractionated on 1.0% agarose gel. The DNA bands were then transferred onto a nylon membrane (Amersham, USA) by upward capillary in a 20× SSC buffer as previously described [ 72 , 73 ]. The HPT DNA was adopted as a probe, and it was prepared from Hind III restricted fragment of pYL-RNAi (Clontech) and labeled with digoxigenin.…”

Using Genetic Engineering Techniques to Develop Banana Cultivars With Fusarium Wilt Resistance and Ideal Plant Architecture

“…Two important aspects of banana molecular breeding are efficient regeneration and genetic transformation ( May et al, 1995 ). In vitro banana cultures have been developed using various explants, such as shoot tips ( Matheka et al, 2019 ), immature male flowers ( Jalil et al, 2003 ), apical meristems ( Novak et al, 1989 ), corms ( May et al, 1995 ), floral apices ( Liu et al, 2016 ), and ECS ( Vuylsteke and Langhe, 1985 ; Sági et al, 1995 ; Cote et al, 1996 , 2000 ). Bananas are typically genetically transformed using particle bombardment or Agrobacterium -mediated transformation ( May et al, 1995 ; Sági et al, 1995 ).…”

Genetic Transformation of Arachis hypogaea Using Novel Genes Conferring Fungal Resistance-A Review

“…There is scope to improve more peanut cultivars and other legumes transformation by using these compounds. Measures like explant wounding /micro-injury have been widely followed in transformation studies where some of them have reported a low frequency of transient transformation (116,147). As an alternative to mechanical wounding, sonicationinduced tissue wounding or whisker-mediated transformation can be tried to increase the transformation frequency (113,146).…”