An Efficient Genetic Transformation and CRISPR/Cas9-Based Genome Editing System for Moso Bamboo (Phyllostachys edulis).

Biyun Huang,Renying Zhuo,Jing Xu,Yujun Wang,Huijin Fan,Kangming Jin,Guirong Qiao

doi:10.3389/fpls.2022.822022

Biyun Huang, Renying Zhuo + Show 5 more

Open Access

https://doi.org/10.3389/fpls.2022.822022

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Abstract

Moso bamboo (Phyllostachys edulis) is the most important monopodial bamboo species worldwide. Without a genetic transformation system, it is difficult to verify the functions of genes controlling important traits and conduct molecular breeding in moso bamboo. Here, we established a plant regeneration system from immature embryos. Calli were induced on MS medium added 4–6 mg⋅L–1 2,4-dichlorophenoxyacetic acid (2,4-D) with high efficiency (>60%). A plant growth regulator combination of 0.5 mg⋅L–1 1-naphthylacetic acid (NAA), 2.0 mg⋅L–1 6-benzylaminopurine (BAP), and 3.0 mg⋅L–1 zeatin (ZT) was suitable for shoot differentiation, and the shoot induction frequency was increased to 43% after 0.5 mg⋅L–1 abscisic acid (ABA) pretreatment. An effective antibiotic screening concentration was determined by hygromycin sensitivity test. We further optimized the Agrobacterium concentration and added vacuum infiltration for infection, which improves the transient expression efficiency. A genetic transformation system was established for the first time in moso bamboo, with the transformation efficiency of approximately 5%. To optimize genome editing, two endogenous U3 small nuclear RNA (snRNA) promoters were isolated and used to drive small guide RNA (sgRNA) expression. The results showed that the PeU3.1 promoter exhibited higher efficiency, and it was used for subsequent genome editing. Finally, homozygous pds1pds2 mutants were obtained by an efficient CRISPR/Cas9 genome-editing system. These technical systems will be conducive to gene functional validation and accelerate the molecular breeding process of moso bamboo.

Highlights

Bamboo is an important forestry resource with a global bamboo forest area of more than 47 million hm2 and an annual output of more than 500 million tons of bamboo wood (Li and Fu, 2021)
Genetic engineering technology has been successfully applied to many plant species, but the lack of efficient regeneration system is the major bottleneck of its application in bamboo
Plant regeneration systems for bamboo have mostly been reported in sympodial bamboo species

Summary

Introduction

Bamboo is an important forestry resource with a global bamboo forest area of more than 47 million hm and an annual output of more than 500 million tons of bamboo wood (Li and Fu, 2021). Moso bamboo (Phyllostachys edulis) is the most economically important woody bamboo species and plays essential economic and ecological roles. This species is characterized by asexual reproduction, rapid growth, and a high carbon fixation capacity (Zhou et al, 2011). Genetic Transformation of Moso Bamboo has revealed the low genetic diversity of the moso bamboo population, which indicates that this species may have a low effective population size and a small genetic pool that can be used for future breeding purposes (Zhao et al, 2021). Due to the long and unpredictable flowering cycle of bamboo, it is difficult to obtain new varieties through crossbreeding. Identifying the genes controlling important bamboo traits and using molecular breeding to improve varieties are the methods that can be used to obtain new varieties quickly

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