Cannabis sativa L. - Botany and Biotechnology

(Jacob Rumans) #1

plant tissue culture or specialized regeneration protocols or equipment. Therefore,
results can be obtained in a much shorter time than having to generate stable
transgenic plants (Sparkes et al. 2006 ). Consequently, agroinfiltration has the
advantage of being a faster and more convenient technique to evaluate gene
expression in plants.


16.2.3 Hairy Root Transformation Using


Agrobacterium rhizogenes


TheA. rhizogenesRi-plasmid harbors T-DNA encodingrootoncogenicloci(rol)
genes that are responsible for the production of hairy roots - highly branched, fast
growing roots covered in root hairs (Veena and Taylor 2007 ). Hairy roots grow out
of the infection site and can be cultured in vitro or hosted by plants with
untransformed aerial tissue (Ron et al. 2014 ). An attractive characteristic of hairy
root cultures is their enhanced ability to synthesize secondary metabolites
(Srivastava and Srivastava 2007 ; Mathur et al. 2010 ). In addition, genes of interest
can be cloned into a T-DNA region on a binary vector and introduced into
A. rhizogenesfor plant transformation (Zhang et al. 2004 ; Ron et al. 2014 ; Sun et al.
2015 ). Thus, hairy roots are a valuable tool for applications such as the study of
biochemical pathways, gene expression, recombinant protein production and
metabolic engineering (Zhang et al. 2004 ; Ono and Tian 2011 ; Ron et al. 2014 ).


16.3 Genetic Transformation ofCannabis sativa


C. sativais amenable to transformation withAgrobacteriumspecies. Two previ-
ously published reports have demonstrated stable transformation of Cannabis.
A. tumefacienswas used to transform cell suspension cultures of the seed cultivar
Anka with a selectable marker gene (Feeney and Punja 2003 , 2015 ). However,
plants could not be regenerated from stable transformed cells in tissue culture.
Subsequently, Wahby et al. ( 2012 ) established procedures to generate stable
transformed tumor and hairy root lines of threefibre and two drug cultivars using
severalA. tumefaciens andA. rhizogenes wild type strains, respectively. The
technology has only rarely been applied toward improvement of Cannabis with
desirable traits. In the only report of which we are aware, MacKinnon et al. ( 2000 )
developed transgenic hemp plants from twofibre cultivars, Fedora 19 and Felina
34, that were resistant toBotrytis cinerea.A. tumefacienswas used to transform
hemp shoot tip explants with genes encoding polygalacturase inhibiting proteins,
conferring resistance to the fungal pathogen. Other methods to transformC. sativa
have not been reported in the published literature.


16 The Role ofAgrobacterium-Mediated and Other Gene-Transfer... 347

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