Establishment and optimization of agrobacterium-mediated transformation in blueberry (Vaccinium species)

Highlights

• The combination of two mg/l zeatin and one mg/l indole-3-butyric acid is critical for the regeneration of Vaccinium leaves.

• The diploid Vaccinium reticulatum ‘Red Button’ has the highest efficiency of callus formation and adventitious bud regeneration.

• Agrobacterium-mediated transformation efficiency in ‘Red Button’ is about 12.82%.

Abstract

Vaccinium species such as blueberry, cranberry and bilberry are widely planted in the world and have high economic values. Current varieties are mostly generated by conventional breeding methods, and application of biotechnology especially genetic modification in the breeding process is seldom. Moreover, mechanistic studies of orphan genes in Vaccinium species are more and more important to facilitate the improvement of yield, quality, resistance to abiotic and biotic stresses in breeding, which requires efficient genetic transformation systems. In this study, we select 19 Vaccinium varieties to investigate the effects of variety specificity, explant types and culture schemes on callus development, adventitious bud regeneration and transformation efficiency. The wood plant medium (WPM) contains sufficient nutrients for the effective callus induction from mature leaves of plants grown in the field. Both zeatin and indole-3-butyric acid with the concentration of 2.0 mg L⁻¹ and 1.0 mg L⁻¹, respectively, are critical for adventitious bud regeneration from a variety of Vaccinium leaves. Different Vaccinium varieties have distinct regeneration efficiencies from leaf discs, and the diploid wild Vaccinium reticulatum ‘Red Button’ has the highest efficiency of callus formation and adventitious bud regeneration. Furthermore, we establish an efficient Agrobacterium-mediated genetic transformation system using ‘Red Button’, in which the transformation efficiency of green fluorescent protein (GFP) encoding gene is about 12.82%. Taken together, our study establishes an efficient tissue culture and regeneration system from leaves of Vaccinium varieties, and identify the diploid material ‘Red Button’ as an ideal variety for genetic transformation, which would greatly accelerate the mechanistic studies and breeding program of Vaccinium genus using emerging plant biotechnologies in the future.

Introduction

Vaccinium (Vaccinium spp.), a shrub genus of Ericaceae (Rhododendron), is composed of 450 species in 33 types (Kulkarni et al., 2020). Blueberry, cranberry and bilberry are three major Vaccinium species with large production and high economic value, and widely planted in the world (Jaakola et al., 2002; Rowland et al., 2012; Li et al., 2017; Samkumar et al., 2021). Vaccinium fruits are rich in proanthocyadin, flavonoids and other physiological active substances of polyphenols, which have health benefits such as improving immunity, resisting cardiovascular diseases and delaying aging (Liu et al., 2010; Cardenosa et al., 2016; Li et al., 2021; Samkumar et al., 2021; Trivedi et al., 2021).

In the past decades, conventional breeding significantly improved economic traits of Vaccinium varieties including higher yields, larger fruits, better shape and quality (Ehlenfeldt and Prior, 2001; Diaz-Garcia et al., 2020; Kulkarni et al., 2020). Most of Vaccinium cultivars are tetraploid or hexaploid , and originally domesticated in North America. The genetic diversities of these cultivars were dramatically reduced by natural and artificial selection during the conventional breeding (Brevis et al., 2008; Gupta et al., 2015), which raises the risks facing the emerging abiotic and biotic stresses. In addition, high genomic heterozygosity, strong progeny separation and inbreeding recession in current Vaccinium varieties hinder the generation of new varieties in a short period through the conventional breeding (Ferrão et al., 2018).

Biotechnology has been widely adopted in the crop breeding, in which introduction of exogenous genes improves agronomic traits including yield, quality and resistance to abiotic and biotic stresses, and accelerates the generation of new varieties (Charrier et al., 2019; Song et al., 2019; Zhang et al., 2021). Effective tissue culture and regeneration is the basis for the application of biotechnology, in which woody plant medium (WPM) supplemented with zeatin (ZT) and indole-3-butyric acid (IBA) is utilized for the callus induction and regeneration using Vaccinium leaves (Qiu et al., 2018). Previous study reported that the regeneration efficiency using leaf discs differed greatly in different highbush Vaccinium cultivars, which was attributed to genetic difference (Liu et al., 2010). The genetic transformation with GUS reporter gene succeeded in four highbush Vaccinium varieties, while the transformation efficiency varied (Song and Sink, 2004). Since then, Bar and CBF gene were transformed into Vaccinium varieties to improve the resistance against herbicide and cols stress, respectively, (Walworth et al., 2012; Song et al., 2007; Song and Walworth, 2018).

Agrobacterium-mediated transformation has been successfully conducted using leaf discs in Vaccinium varieties, while the adaptability and efficiency of methods developed in previous studies need to be investigated and improved. Polyploid and heterozygosity in Vaccinium varieties impede the efficiency of genetic transformation and functional studies of transgenes. An efficient regeneration system for Vaccinium varieties will assist the application of biotechnology in Vaccinium breeding, and accelerate the generation and spreading of new cultivars. A highly efficient transformation system under a uniform genetic background will boost functional studies of orphan genes in Vaccinium varieties. In this study, we optimize the concentration of plant hormone ZT and IBA for efficient regeneration of various Vaccinium varieties, and establish a sophisticated procedure for tissue culture and regeneration using Vaccinium leaf discs. We also investigate the efficiency of regeneration in multiple Vaccinium varieties, and identify a diploid Vaccinium reticulatum cultivar ‘Red Button’ which has highest regeneration efficiency with shortest time compared to other varieties. We further utilize ‘Red Button’ to successfully develop a robust Agrobacterium-mediated genetic transformation system. Taken together, our results provide tools to propel the basic research in Vaccinium varieties, and accelerate the application of biotechnology such as gene editing technology in Vaccinium breeding.