Efficient mutation induction using heavy-ion beam irradiation and simple genomic screening with random primers in taro (Colocasia esculenta L. Schott)

Highlights

• Multiple shoots of the taro cultivar were subjected to heavy-ion beam irradiation.

• A high survival rate for genomic screening was achieved following irradiation.

• Genomic screening by random primers revealed polymorphic DNA patterns.

• A single dose of irradiation generated mutants without somatic mosaicism.

• The polymorphic patterns would facilitate intra- and inter-cultivar identification.

Abstract

Progress in conventional breeding methods for taro (Colocasia esculenta L. Schott) via crossing has been limited, and suitable genetic materials for the development of new cultivars are scarce as most commercial taro cultivars are either non-flowering or rarely flowering triploids. In an attempt to advance taro breeding, we performed mutational breeding by heavy-ion beam irradiation of multiple shoots of ‘Chiba maru’ cultivar. Using 2–10 Gy neon and carbon ion beams, we achieved a plant survival rate of more than 90 % and used 94 surviving plants for genomic screening. To efficiently detect DNA polymorphisms induced by ion beam irradiation in young plants, we used five sets of 15-mer randomly amplified polymorphic DNA and arbitrarily primed-polymerase chain reaction random primers based on retrotransposon sequences for genomic screening. Two plants had polymorphic DNA bands, and the specific DNA patterns were maintained in all leaves. In one of these plants, which lacked somatic mosaicism (Cm10), the polymorphic patterns were maintained in the leaves and cormels of clones propagated from daughter cormels. Ion beam irradiation of multiple taro shoots could thus generate mutants that can be developed as new cultivars; the resulting novel polymorphic patterns would facilitate inter- and intra-cultivar identification.

Introduction

Taro (Colocasia esculenta L. Schott) is widely distributed in tropical and sub-tropical areas, mainly in Oceania, Asia, and Africa, and is one of the most important staple food crops in the Pacific Islands (Matsuda and Nawata, 2002; Chaïr et al., 2016). In Japan, taro has a long history of cultivation, as its corms are a good source of protein, calcium, and phosphorus (Kumazawa et al., 1956). In 2017, Japan produced 148,600 tons of taro, 10.9 % of which was grown in Chiba Prefecture, one of Japan’s foremost agricultural regions (https://www.e-stat.go.jp). However, to date, the progress of conventional breeding of taro by crossing is limited, and suitable materials to generate new cultivars are scarce because most commercial taro cultivars are triploid and rarely flower (Kumazawa et al., 1956; Chaïr et al., 2016). Consequently, the Chiba Prefectural Agriculture and Forestry Research Center has undertaken soft X-ray-based mutational breeding using ‘Dotare’ as the original cultivar, generating the new cultivar, ‘Chiba maru’ with round corms, facilitating commercial processing and sorting using auto-selection machines (Suzuki et al., 2006). The cultivar has also been modified to confer additional valuable traits, nutritive value, and disease resistance by further breeding using various physical mutagenic and novel micropropagation techniques (Nakagawa and Kato, 2017).

Ion beams (protons and carbon and neon ions) have been used extensively to examine the tolerance of materials to cosmic rays in space science research and in general DNA mutation and repair. They have higher average energy transferred per unit length of track during radiation (termed linear energy transfer [LET]) than conventional irradiation techniques, facilitating penetration to deep-seated cells and tissues. This generates point mutations and rearrangements (including deletion and homologous recombination induced by double strand break repair) within cells. The use of heavy ion beams such as carbon- and neon-ion beams facilitates the strict control of irradiation intensity on deep-seated targets (Bragg-peak) and the areas irradiated, and it has been applied in cancer therapy, biological examinations, and plant breeding (Shikazono et al., 2005; Ichida et al., 2008; Kamada et al., 2015; Sasaki et al., 2018). In the present study, we used neon and carbon-ion beams as physical mutagens to induce mutations in taro.

We previously reported the identification of new chrysanthemum cultivars (Chrysanthemum morifolium Ramat. ‘Jimba’) induced by carbon-ion beam irradiation using a leaf disk regeneration system; we performed polymerase chain reaction (PCR)-based genomic screening using random primers to develop DNA markers for cultivar identification (Shirao et al., 2013). In taro, a micropropagation system based on multiple shoots derived from apical buds has been reported previously (Suzuki et al., 2016). Here, we used this system in conjunction with heavy-ion beam irradiation for taro, and subsequently performed genomic screening using random primers. This approach, the resulting mutants, and DNA polymorphism data represent a new strategy for mutation breeding as well as DNA marker development for the identification of commercial taro cultivars.