Agrobacterium rhizogenes-dependent production of transformed roots from foliar explants of pepper (Capsicum annuum): a new and efficient tool for functional analysis of genes

Abstract

Pepper is known to be a recalcitrant species to genetic transformation via Agrobacterium tumefaciens. A. rhizogenes-mediated transformation offers an alternative and rapid possibility to study gene functions in roots. In our study, we developed a new and efficient system for A. rhizogenes transformation of the cultivated species Capsicum annuum. Hypocotyls and foliar organs (true leaves and cotyledons) of Yolo Wonder (YW) and Criollo de Morelos 334 (CM334) pepper cultivars were inoculated with the two constructs pBIN-gus and pHKN29-gfp of A. rhizogenes strain A4RS. Foliar explants of both pepper genotypes infected by A4RS-pBIN-gus or A4RS-pHKN29-gfp produced transformed roots. Optimal results were obtained using the combination of the foliar explants with A4RS-pHKN29-gfp. 20.5% of YW foliar explants and 14.6% of CM334 foliar explants inoculated with A4RS-pHKN29-gfp produced at least one root expressing uniform green fluorescent protein. We confirmed by polymerase chain reaction the presence of the rolB and gfp genes in the co-transformed roots ensuring that they integrated both the T-DNA from the Ri plasmid and the reporter gene. We also demonstrated that co-transformed roots of YW and CM334 displayed the same resistance response to Phytophthora capsici than the corresponding untransformed roots. Our novel procedure to produce C. annuum hairy roots will thus support the functional analysis of potential resistance genes involved in pepper P. capsici interaction.

Alternative method for the transformation of Capsicum species

“…In this study, we are trying to achieve an ef icient way for validating genes involved in resistance against soil pathogens and genes which take part in the root nodulation and formation. Our research was based on the work of Aarrouf, et al 2012, with modi ications in the plant variety and inoculation method [14]. The development of "hairy root" phenotype (Figure 1) in A. rhizogenes mediated Capsicum transformants showed a thin ilamentous structure (similar to the mycelia in fungi) especially along the surface of the cutting and pricked sites, however, not all transformant showed hairy root phenotype.…”

Cloning, Characterization and Functional Analysis of Two Type 1 diacylglycerol acyltransferases (DGAT1s) from Tetraena mongolica

“…In the present study, transgenic soybean hairy roots and transgenic T. mongolica calli were used as two quick and efficient systems for TmDGAT1s function verification, which offer rapid techniques to introduce foreign genes in plant cells (Aarrouf et al 2011). Production of transgenic hairy roots with A. rhizogenes only takes a few weeks (Guillon et al 2006).…”

Agrobacterium rhizogenes-Mediated Transformation and Its Biotechnological Applications in Crops

“…In general, hairy root cultures have been used extensively in root nodule research (Diaz et al 1989;Quandt et al 1993;Diouf et al 1995;Hu and Du 2006;Hirotaka and Hiroshi 2003;Aarrouf et al 2012), production of artificial seeds (Uozumi and Kobayashi 1997), plant secondary metabolites and proteins (Aarrouf et al 2012), plant breeding and plant improvement, experimental systems to study responses to chemicals (Downs et al 1994;Mugnier 1997), plant morphology and development (Bandyopadhyay et al 2007; Turgut- Kara and Ari 2008;Hasancebi et al 2011;Aarrouf et al 2012), detoxifing environmental pollutants (Rugh 2001), validate and analyze the functions of genes conferring resistance to root specific pathogens (Remeeus et al 1998;Hwang et al 2000;Alpizar et al 2006;Aarrouf et al 2012) and study interactions with other organisms such as nematodes (Kifle et al 1999), mycorrhizal fungi and root pathogens (Mugnier 1997;Christey 2001). Besides these sights, enhanced rooting in plants helps establishment or surviving transplant shocks or abiotic stress like drought, salinity and heavy metal stress (Bulgakov, 2008;Li et al 2011).…”

“…It is also known that modification of the cell hormonal balances occurring in response to infection causes root formation at the infected site (Gaudin et al 1994;Aarrouf et al 2012). However, the response varies depending upon the strain and its interaction with the plant.…”