Establishment of an Agrobacterium-mediated genetic transformation and CRISPR/Cas9-mediated targeted mutagenesis in Hemp (Cannabis Sativa L.)

Abstract

Hemp (Cannabis sativa L.) is an annual and typically dioecious crop. Due to the therapeutic potential for human diseases, phytocannabinoids as a medical therapy is getting more attention recently. Several candidate genes involved in cannabinoid biosynthesis have been elucidated using omics analysis. However, the gene function was not fully validated due to few reports of stable transformation for Cannabis tissues. In this study, we firstly report the successful generation of gene-edited plants using an Agrobacterium-mediated transformation method in C. sativa. DMG278 achieved the highest shoot induction rate, which was selected as the model strain for transformation. By overexpressing the cannabis developmental regulator chimera in the embryo hypocotyls of immature grains, the shoot regeneration efficiency was substantially increased. We used CRISPR/Cas9 technology to edit the phytoene desaturase gene and finally generated four edited cannabis seedlings with albino phenotype. Moreover, we propagated the transgenic plants and validated the stable integration of T-DNA in cannabis genome.

Keywords: Agrobacterium-mediated transformation; CRISPR; Cas9; Hemp; genome editing.

Figure 1

Callus induction and shoot regeneration from different explants of YUNMA7. The developmental stages are described as follows: (a, b and c) Isolated true leaves, cotyledons and hypocotyls from 17‐days‐old seedlings. (d) Embryo hypocotyls dissected from immature grains 15 days after anthesis; co: cotyledon; hy: hypocotyl. (e, f, g and h) Formation of embryogenic calli at the callus induction medium after 29 days of in vitro culture. (i, j, k and l) Development of shoots at the regeneration medium after 78 days of in vitro culture. Scale bars: 2 cm.

Figure 2

Effect of cannabis varieties on the shoot regeneration rate of immature embryo hypocotyls. For each variety, 90–130 hypocotyls were dissected from immature embryos, cultured in callus induction medium and then transferred to regeneration medium. The regeneration rate was calculated as the percentage of regenerated shoots /total calli for each variety, which were 7.09% for DMG278 and 6.12% for YUNMA7. The two varieties are coloured red and yellow.

Figure 3

Effect of developmental regulators on shoot regeneration frequency. All the calli used in the transformation experiments were produced from the hypocotyls of immature DMG278 grains (15 days after anthesis). Box plots show the regeneration frequencies for different regulator combinations as well as the control (empty pKSE401 vector). The box is divided by the median with the range from the first to third quartile, and the range of whiskers from the minimum to the maximum values in each treatment (***P < 0.001). The growing green shoots are noted in the callus (red arrows). Control: pKSE401 empty vector.

Figure 4

Indel efficiency of pG41sg at the six targeting sites of CsPDS1. The empty pG41sg vector was used as a control. The indel frequency was calculated as the percentage of successful mutations for each target sites, which were 7.55% for guide3, 3.43% for guide4 and 4.72% for guide5.

Figure 5

Representative images of gene‐edited shoots and sequencing results of indels at the desired place. (a) A gene‐edited shoot germinated from the callus that displayed photobleaching. (b and c) Chimeric tissues with the upper shoot green and lower shoot white (red arrows) as well as white shoots in developmental abnormalities (white arrows). (d) and (e) Sequencing results of indels at the desired place and their chromatograms. The black arrows indicate the mutations. The red line denotes the guide3‐sgRNA target sequence. The blue line denotes the protospacer adjacent motif (PAM) sequence serves as a binding signal for Cas9.

Figure 6

Transgenic cannabis seedlings and results of transgenic screening. (a) Shoots regenerated from stems of the transgenic seedling. In the estimation of developmental regulator effects on shoot organogenesis, we obtained one transgenic seedling G41‐1 carrying the pG41sg T‐DNA fragment. Then G41‐1 stem was cut into pieces and incubated in the regeneration medium containing kanamycin for 6 weeks. There are five shoots germinated from the stem explant. (b) A transgenic seedling regenerated from the G41‐1 stem. All the five shoots were transferred to soil after a 5‐week incubation in the root‐induction medium. The first fully expanded leaves were sampled every three weeks when growing in greenhouse. Red circle: sampling in the first round of screening; white circle: sampling in the second round of screening. (c) Transgenic‐specific PCR result of the chimeric plants containing mutagenesis at CsPDS1. Eleven chimeric seedlings (chim1‐11) were randomly selected and transferred to soil after incubation in the root‐induction medium. Since their first fully expanded leaves lost the T‐DNA fragment, these plants were identified as no transgenic with primers AtU6‐F1/R1 in the second round of screening. P: DNA sample of pG41sg, N: DNA sample of no transgenic plant; white arrows: specific PCR product. (d) and (e) Transgenic‐specific PCR results of the five seedlings regenerated from G41‐1. In the second round of screening, these plants (Cas9‐1 to Cas 9‐5) were identified as transgenic plants based on transgenic‐specific PCR results amplified with primers AtU6‐F1/R1 and CsCAS9F2/R2.