Efficient Biolistic Transformation of Immature Citrus Rootstocks Using Phosphomannose-isomerase Selection.

Wu Wu,Canton Canton,Zale Zale,Acanda Acanda

doi:10.3390/plants8100390

Wu Wu, Canton Canton + Show 2 more

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https://doi.org/10.3390/plants8100390

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Abstract

This research utilized the E. coli manA gene encoding phosphomannose isomerase (PMI) selection on sucrose/mannose medium to increase transformation efficiencies after biolistic transformation of two immature citrus rootstock cultivars. Plasmid DNA, containing the manA gene and the enhanced green fluorescent protein (egfp) reporter gene, was bombarded into epicotyl explants of immature Carrizo citrange and Swingle citrumelo. GFP positive shoots were micro-grafted onto in vitro grown immature Carrizo rootstocks. Nineteen transgenic Carrizo shoots were obtained from ten paired shots, and eight Swingle shoots from five paired shots. The mean transformation efficiency of Carrizo was 1.9 transgenics/paired shot while the transformation efficiency of Swingle was comparable at 1.6 transgenics/paired shot. The transformants were analyzed by PCR for the presence of transgenes. Southern blot analysis of eight representative Carrizo transgenic events and four Swingle transgenic events showed that all transgenics had one to three copies of the manA gene. The PMI enzyme activity in the transgenic lines was confirmed using the chlorophenol red assay.

Highlights

For the 2016–2017 season, citrus production in Florida was drastically reduced due to Huanglongbing (HLB), known as citrus greening disease, and Hurricane Irma
E. coli phosphomannose-isomerase (PMI) enzyme activity is encoded by the manA gene and it has been used as a selectable marker in the genetic transformation of several crops, such as almond (Prunus dulcis ) [3], apple (Malus domestica Borkh.) [4], cassava (Manihot esculenta Crantz) [5], maize
Five experiments, comprised of ten paired shots, were carried out with immature Carrizo citrange rootstock explants to determine the efficacy of phosphomannose isomerase (PMI) in the selection of transgenic shoots after biolistic transformation

Summary

Introduction

For the 2016–2017 season, citrus production in Florida was drastically reduced due to Huanglongbing (HLB), known as citrus greening disease, and Hurricane Irma. Total citrus production was 78,130 million boxes (40.8 kilograms/box), which was down from 291,800 million boxes during the 2003–2004 season [1]. Citrus improvement is complicated by nucellar embryony, which makes the production and selection of hybrids difficult, and by the long juvenile phase, which can last 3 to 20 years. The 2018 National Academy of Sciences report recommended the production of transgenics and gene-edited plants to impart disease resistance because it might be faster than traditional breeding [2]. L.) [6], rice (Oryza sativa L.) [7], and wheat (Triticum aestivum L.) [6,8].

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