Establishment and optimization of agrobacterium-mediated transformation in blueberry (Vaccinium species)☆
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.
Section snippets
Plant materials
Nineteen Vaccinium varieties used in this study were planted in the filed with regular irrigation and fertilization. Descriptions of these varieties including Reticulatum, Lowbush, Highbush and Rabbiteye were list in Supplementary Table 1. All plants are turn green and bloom in the spring.
Explant and media preparation
The young tip leaves of about 2-week-old healthy and pest-free plants from cuttings grown in the field were selected. The surface was wiped repeatedly with detergent and cleaned thoroughly with tap water for 1
The concentrations of plant hormones ZT and IBA are important for callus development in Vaccinium varieties
In order to obtain highly efficient callus induction and differentiation system, we investigated the effects of sterilization procedure of explants and the concentration of plant hormone ZT and IBA. We harvested fully expanded leaves of highbush Vaccinium variety 'Legacy' grown in the field, and sterilized them with 75% alcohol for 45s, and then treated with 8% sodium hypochlorite as previously described. The combination of alcohol and sodium hypochlorite could effectively remove the bacteria
Discussion
Biotechnologies are widely applied in the plant breeding, and especially new biotechnology such as Crispr-Cas based gene editing technology would be adopted to generate new varieties in the near future. Lack of functional studies of Vaccinium genes hinder the application of biotechnology in Vaccinium breeding, which requires a model Vaccinium specie with a highly efficient genetic transformation system. In this study, we optimize the concentration of plant hormones ZT and IBA, and compare
Conclusion
Our study compares the efficiency of callus initiation, growth and differentiation in various Vaccinium varieties and establishes a highly efficient Agrobacterium-mediated genetic transformation system for studying Vaccinium gene functions. Plant hormones ZT and IBA at the concentration of 2.0 mg L−1 and 1.0 mg L−1, respectively, can effectively stimulates the callus initiation, growth and differentiation of Vaccinium varieties using leaf discs. The diploid variety ‘Red Button’ has the highest
Funding information
This work was supported by the National Key Research and Development Program of China (2019YFD1000101) and Taishan Scholar Foundation of Shandong Province (tsqn201909073).
CRediT authorship contribution statement
Guodong Wang: Formal analysis, Investigation. Minmin Liu: Formal analysis, Investigation. Genzhong Liu: Resources. Zhilong Bao: Supervision, Project administration, Writing – review & editing. Fangfang Ma: Project administration, Writing – review & editing, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This research was supported by National Key Research and Development Program of China (2019YFD1000101 to F.M.), Taishan Scholar Foundation of Shandong Province (tsqn201909073) and Startup funding from Shandong Agricultural University for Dr. Fangfang Ma and Dr. Zhilong Bao. We thank Dr. Yadong Li and Genzhu Zhang to provide Vaccinium materials. We thank plant growth facility members in the state key laboratory of crop biology at Shandong Agricultural University.
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