Agrobacterium-mediated genetic transformation of tea leaf explants: effects of counteracting bactericidity of leaf polyphenols without loss of bacterial virulence

Abstract

Tea is one of the major crops in Asia and Africa, and its improvement by genetic modification is important for economy of many tea-producing regions. Although somatic embryos derived from cotyledon explants have been transformed with Agrobacterium, the leaves of several commercially important tea cultivars have remained recalcitrant to transformation, largely due to bactericidal effect of polyphenols that are exuded by tea leaves in vitro. Moreover, the commonly used polyphenol adsorbents and antioxidants cannot overcome this problem. Leaf explants, however, are more desirable than cotyledon-derived somatic embryos, especially when it is necessary to further improve a selected elite and also retain its superior traits. Thus, we developed a procedure for Agrobacterium-mediated genetic transformation of tea leaf explants which is based on the presence of L-glutamine in the co-cultivation medium. We then showed that the transformation process is facilitated via a protective action of L-glutamine against bactericidal effects of leaf polyphenols without affecting the bacterial virulence (vir) gene expression.

Producing low-caffeine tea through post-transcriptional silencing of caffeine synthase mRNA

“…Further, the RNAi based on hpRNA strategy has been reported as more efficient for gene silencing (Smith et al 2000). Also methods are available for transformation and regeneration in tea (Lopez et al 2004;Bhattacharya et al 2006;Sandal et al 2007). Our recent study has documented the regulatory role of caffeine synthase (CS) in caffeine biosynthesis of tea (Mohanpuria et al 2009).…”

“…It was used successfully in the production of first healthy transgenic tea plants via Agrobacterium-mediated transformation (Mondal et al 2001a). Further, tea transformations and production of transgenics have been attempted via Agrobacterium or biolistic mediated approach by several workers using different explants such as embryogenic tissues, somatic embryos, and in vitro leaves (Lopez et al 2004;Jeyaramraja and Meenakshi 2005;Bhattacharya et al 2006;Sandal et al 2007). …”

Genetic transformation of gentian Gentiana tibetica (King) leaf explants with Agrobacterium tumefaciens strain C58C1

“…Here are a few examples: 10 min of incubation and 3 days of co-cultivation for Drosera rotundifolia (Hirsikorpi et al 2002), 15 min for incubation and 4-day-long co-cultivation for Plumbago zeylanica (Wei et al 2006), 20 min of incubation and 5-day-long co-cultivation Camellia sinensis (Sandal et al 2007), and 30 min of incubation and 4-daylong co-cultivation for Coffea canephora (Hatanaka et al 1999). It should be emphasized that cell suspension, regardless of its type, requires a considerably longer time of the bacteria incubation, for example: Arabidopsis thaliana 48 h (Gallego et al 1999) and Nicotiana tabacum even 72 h (Rempel and Nelson 1995).…”

“…It acts into two fields of relation between the bacteria and plant cell. L-Glutamine affects the effectiveness of transformation by protection of the bacterial cell against plant polyphenols and protects bacterial abilities to transfer the fragment of T-DNA (Sandal et al 2007) into a plant cell. Very efficient Agrobacterium-mediated transformation of the liverwort Marchantia polymorpha thalli was developed with the help of L-glutamine used for co-cultivation medium (Kubota et al 2013).…”

“…L-Glutamine effect on transformation efficiency L-Glutamine is the one of the organic substances tested for improving the vector transformation efficiency for monocots (Kubota et al 2013) and for plant species with very rich secondary metabolites; Camellia sinensis (Sandal et al 2007). It acts into two fields of relation between the bacteria and plant cell.…”