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

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.

Data-Independent Acquisition Proteomics Reveals the Effects of Red and Blue Light on the Growth and Development of Moso Bamboo (Phyllostachys edulis) Seedlings

“…Although a genetic transformation system for moso bamboo has recently been established [ 22 ], the long vegetative phase of moso bamboo makes it difficult to obtain transgenic seeds, leading to difficulty in applying traditional genetic methods to study the molecular mechanisms of photomorphogenesis in moso bamboo seedlings after seed germination. Therefore, omics has become an effective method for studying the potential molecular mechanisms underlying the effects of different light qualities on the growth of bamboo.…”

A new biotechnology for in-planta gene editing and its application in promoting flavonoid biosynthesis in bamboo leaves

“…In order to speed up the breeding process, people try to use modern biotechnology to carry out a lot of research work, such as space breeding, mutation breeding, and genetic engineering breeding. Recently, genetic transformation and gene editing systems have been reported in bamboo [ 3 – 5 ], and all of them are using callus for transformation through Agrobacterium . However, the callus induction and regeneration process is time-consuming, labor-intensive, and complicated.…”

“…Bamboo leaf flavonoids have a variety of activities such as antioxidant, anti-inflammatory, anti-ulcer, and antitumor, which can help reduce the risk of cardiovascular disease, Alzheimer’s disease, Parkinson’s disease, and diabetes in previous studies [ 1 , 2 ]. Genetic transformation and gene editing systems in bamboo have been established via callus induction [ 3 – 5 ]. However, the application of the existing gene editing systems in bamboo is primarily restricted by the dependency on the regeneration capability of bamboo, which is highly time-consuming (lasting usually over one year), labor-intensive, and complicated.…”

RNA Pol III promoters—key players in precisely targeted plant genome editing

“…The first report of an immature embryo plant regeneration system and genetic transformation system in Phyllostachys edulis , a monopodial bamboo species using two PeU3 promoters and targeting the PePDS1 and PePDS2 genes. The usage of endogenous pol III promoters led to higher editing efficiency (35%–39%) than editing with the OsU3 promoter ( Huang et al, 2022 ). White spruce is one of the major sources of timber and pulpwood, having high economic and ecological importance.…”