Abstract
In the process of acquiring mutants mediated by CRISPR/Cas9, plantlets are often regenerated from both mutated and non-mutated cells in a random manner, which increase the odds of chimeric mutated plant. In general, it’s necessary to infect more explants or grow to next generation for the need of generating more biallelic or homozygous mutants. In present study, an efficient way of obtaining biallelic or homozygous mutated lines via fast-growing hairy root system without increasing numbers of infected explants or prolonging sexual propagation generation is reported. The fast growing lateral branches of hair roots are originated deep within the parental root from a small number of founder cells at the periphery, and therefore were employed as a library that classify different editing types in different lateral branches in which the homozygous or biallelic lines were screened. Here, MtPDS was employed in a proof-of-concept experiment to evaluate the efficiency of genome editing with our hairy root system. Homozygous/biallelic mutations were found only 1 of the 20 lines in the 1st generation hairy roots, and 8 lines randomly selected were cultured to obtain their branch roots, homozygous/biallelic mutations were found in 6 of the 8 lines in their branch roots. We also tested the method with MtCOMT gene and got the same result. All of the seedlings regenerated from the homozygous/biallelic hairy root mutation lines of MtPDS displayed albino phenotypes. The entire process from vector design to the recovery of plantlets with homozygous/biallelic mutations took approximately 4.5–6.5 months. The whole process could bring inspiration for efficiently generating homozygous/biallelic mutants through CRISPR/Cas9 system from the hairy root or root system of a chimeric mutated transformants, especially for the rare and endangered plants whose explants sources are very limited or the plants that lack of tissue culture and rapid propagation system.
Highlights
The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system is being harnessed as a powerful tool for gene engineering in crop improvement (Xie et al, 2015; Shah et al, 2018)
Our results indicate that the numerous CRISPR/Cas9mediated homozygous/biallelic mutants can be obtained in one month after two rounds of hairy root culture
We found that of all the A. rhizogenes strains tested, LBA9402 exhibited the highest hairy root induction rate (Supplementary Table S1)
Summary
The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system is being harnessed as a powerful tool for gene engineering in crop improvement (Xie et al, 2015; Shah et al, 2018). As long as the resistance gene sequence is integrated into the plant genome and is expressed normally, transformed plants, tissues or calli can survive selection and grow regardless of whether the target sequences are edited by the CRISPR-Cas system or not This results in the recovery of plants containing both mutated and non-mutated cells in a random manner, and increases the odds of recovering chimeric mutated plants (Nishitani et al, 2016; Pan et al, 2016). The fact that only a small number of founder cells at the periphery of the parental root could be inherited and transferred their genetic materials in new formed lateral branches, but not retain all the calli as the situation in rice (Gibbs and Coates, 2014) This made us realize that the fast-growing branches of hairy roots could be considered a library in which different editing types produced by the CRISPR/Cas genome-editing system can be classified in different lateral branches. This hairy root selection system can facilitate a highly efficient generation of homozygous/biallelic mutants for genome engineering of forage and other agriculturally important crops
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