Effects Of Catechins On Agrobacterium-Mediated Gene Transformation Of Camellia Sinensis

In this study, recalcitrance of tea plant (Camellia sinensis) to Agrobacterium-medlated gene transformation was investigated with an emphasis on tea specialized compounds. Tea plant contains abundant secondary metabolic products. The tea plant is often used for studies of plant flavonoid metabolism due to its abundant and diverse flavonoid compounds. Genetic enhancement of beneficial traits and biological studies of tea require a simple and cost-effective approach. Agrobacterium-mediated genetic transformation has been used as a routine lab method for genetic engineering and biological research. However, tea plants are recalcitrant to Agrobacterium-mediated transformation. The biological basis of tea plant recalcitrance is largely unknown and an efficient and reproducible protocol for Agrobacterium-mediated tea plant transformation has not been established, albeit some reports on tea transgenic plants and hairy roots have been published. Tea polyphenols have bacteriostatic activity in previous studies. So the tea polyphenols may affect the physiological activity and plant transformation efficiency of Agrobacterium. The chemical constitutes in tea leaves or calli extracted were extracted using Luria-Bertani (LB) liquid medium and their corresponding authentic standards were examined on Agrobacterium growth and virulence and plant transformation. Compared to control Agrobacterium grown in LB medium containing 6.5 mg/mL of catechins caused agrobacterial growth reduction,73%-36% suppression of the six virulence (vir) gene expressions, and brownness of infected tobacco explants without any events of transient or stable transformation. Tea callus extract containing 0.22 mg/mL catechins did not significantly affect agrobacterial growth or tobacco transgenic hairy root generation but enhanced the expression of some vir genes. Exogenous addition of authentic catechin mixtures, rather than caffeine, in the LB medium resulted in inhibitory effects on agrobacterial growth, vir gene expression, and tobacco transformation as found using the tea leaf extract, suggesting that catechins were the key constitutes in tea leaves. Agrobacterium tumaficiens GV3101 induced limited transient transformation events in tea leaf and callus explants while A. rhizogenes ATCC15834 induced transgenic root generation from the tea explants, ranging from 15% to 20% of root generation rates, with the highest for hypocotyls and cotyledons, followed by leaf and stem sections. Our data suggested catechins inhibition on tea gene transformation could be overcome using adequate Agrobacterium.