Transient expression of the β-glucuronidase gene in Cannabis sativa varieties

Abstract

In plant biology, transient expression analysis plays a vital role to provide a fast method to study the gene of interest. In this study, we report a rapid and efficient method for transient expression in Cannabis sativa seedlings using Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 carrying the pCAMBIA1301 construct with uidA gene was used to transform cannabis seedlings and the GUS assay (a measurement of β-glucuronidase activity) was used to detect the uidA expression. In the current study, we have also established a rapid germination protocol for cannabis seeds. The all three steps seed sterilization, germination and seedlings development were carried out in a 1% H 2 O 2 solution. Transient transformation revealed that both cotyledons and young true leaves are amenable to transformation. Compared with tobacco (Nicotiana benthamiana), cannabis seedlings were less susceptible to transformation with A. tumefaciens. Susceptibility to Agrobacterium transformation also varied with the different cannabis varieties. The method established in this study has the potential to be an important tool for gene function studies and genetic improvement in cannabis.

Advances and Perspectives in Tissue Culture and Genetic Engineering of Cannabis

“…The authors reported that more than 31% of the cells were successfully transformed. Although gene transformation has been achieved in cannabis by different studies [ 141 , 171 , 172 , 173 , 174 , 175 , 176 ], there is only one report regarding transgenic plant regeneration [ 93 ].…”

“…Based on their results, the maximum GUS expression was observed in the CRS-1 genotype and A. tumefaciens GV3101 led to the highest transformation frequency. In another study, Sorokin et al [ 176 ] investigated the potential of A. tumefaciens EHA105 in transforming different cannabis genotypes (Candida CD-1, Holy Grail x CD-1, Green Crack CBD, and Nightingale) and obtained a high transformation efficiency (45–70.6%). Different responses to Agrobacterium strains are not unique to cannabis and it has been previously documented that various Agrobacterium strains differ in their capacity to transform different recalcitrant plants such as maize [ 177 ].…”

“…In another study, Wahby et al [ 173 ] used different parts of 5-day-old in vitro grown seedling of hemp including hypocotyls, cotyledons, cotyledonary node, and primary leaves for gene transformation and reported that the best gene transformation results were obtained from hypocotyl segments. Sorokin et al [ 176 ] also used cotyledons and true leaves of 4-day-old in vitro grown seedling of hemp for gene transformation. Deguchi et al [ 175 ] reported a successful gene transformation using male and female flowers, stem, leaf, and root tissues derived from 2-month-old in vitro grown seedling of hemp.…”

“…In another study, different explants were co-cultured for two days [ 173 ]. Sorokin et al [ 176 ] suggested three days of co-cultivation and OD 600nm 0.6 for gene transformation of different parts of in vitro grown seedling of hemp.…”

“…On the other hand, Deguchi et al [ 175 ] applied 200 μM acetosyringone, 2% glucose, and 10 mM MES for increasing strain virulence in the gene transformation of hemp. Sorokin et al [ 176 ] also reported increasing the Agrobacterium infectability by using 100 µM acetosyringone for cannabis gene transformation in the co-cultivation medium. Recently, Karthik et al [ 178 ] reported that SNP improved the efficiency of Agrobacterium -mediated gene transformation in soybean.…”