CRISPR/Cas9-mediated editing of carotenoid biosynthesis genes alters carotenoid concentrations in kiwifruit

Abstract

Background: CRISPR/Cas9 technology has garnered increasing attention for its simplicity and precision in genome editing, making it an indispensable tool for gene function research and crop genetic improvement. However, the inefficiency and time-consuming nature of genetic transformation continue to pose substantial challenges to its widespread application in woody plants.

Results: In this study, we developed a rapid and efficient Agrobacterium-mediated transformation system using petioles as explants for kiwifruit. Positive resistant seedlings were obtained within three months by inoculating on MS medium supplemented with 2.0 mg·L^-1 6-benzylaminopurine (6-BA), 0.2 mg·L^-1 naphthaleneacetic acid (NAA), and 10 mg·L^-1 hygromycin, which was faster than using leaves as explants. Using this system, CRISPR/Cas9-mediated editing of phytoene desaturase (AcPDS) and ζ-carotene desaturase (AcZDS) achieved an editing efficiency of 20%. Transgenic kiwifruit lines with edited AcZDS exhibited a significant reduction in carotenoid content.

Conclusions: Overall, we established an efficient Agrobacterium-mediated transformation system using petioles as explants, which is applicable for CRISPR/Cas9-mediated gene editing in kiwifruit, thereby facilitating functional gene studies and genetic improvement.

Keywords: ζ-carotene desaturase (ZDS); CRISPR/Cas9; Genetic transformation; Kiwifruit; Phytoene desaturase (PDS).

Fig. 1

The phenotype of regenerating shoots induced on different medium. A, B The petiole induced on M1 and M2 medium; C, D The leaf induced in M1 and M2 medium

Fig. 2

Procedures for genetic transformation of kiwifruit petiole. A Petiole preculture; B Coculture with Agrobacterium; C Antibiotic screening culture; D Cultured in the dark for 14 d; E Bud differentiation induced in the light; F Subculture of resistant buds

Fig. 3

Construction of editing vectors. A Gene structures of AcPDS1 and AcPDS2. T1-T6 were six selected target sites. B CRISPR/Cas9 binary vector structures. Construct of editing vectors was confirmed by PCR production of sgRNA expression cassette

Fig. 4

Phenotype of the obtained regenerated kiwifruit buds transformed with pHSE401-AcPDS. A Growth status of regenerated buds; B Amplification of the pHSE401 vector fragment to confirm positive transgenic kiwifruit lines. M: DL2000 marker; WT: wild type; P1: pHSE-PDS1; P2: pHSE-PDS2; P3: pHSE-PDS3; 1–16: independent transgenic kiwifruit lines

Fig. 5

Sequence analysis of AcPDS editing lines. A The phenotype of editing lines. B Sequencing chromatograms of the mutated target sites. Dashes indicate deletion, target sequences are shown in light blue and the PAM sequence (NGG) is highlighted in yellow

Fig. 6

Positive detection and phenotype of edited kiwifruit lines transformed with pHSE401-AcZDS. A PCR detection of positive transgenic kiwifruit lines. B The phenotype of the editing lines. C Sequencing chromatograms showing mutations at the target site regions

Fig. 7

Sequence analysis of mutants transformed with PTG-AcZDS vector in ‘Hongyang’ kiwifruit. A PCR detection of positive transgenic kiwifruit lines. B The phenotype of editing lines. C Sequencing chromatograms showing mutations at target site regions. Dashes indicate for deletion sites, target sequences are displayed in light blue and the PAM sequence (NGG) is highlighted in yellow

Fig. 8

Carotenoid content in leaves of AcZDS-edited transgenic plants. Data are expressed as mean ± SD with three biological replicates. Significant differences were determined by Student’s t-test (**P < 0.01, n = 3)