Optimizing the transient Fast Agro-mediated Seedling Transformation (FAST) method in Catharanthus roseus seedlings

Abstract

An Agro-mediated transformation method has been adapted in Catharanthus roseus seedlings for transient overexpression. Our results suggest that Agro-mediated methods may induce defense-related genes, which should be considered in its application. The Fast Agro-mediated Seedling Transformation (FAST) method, which involves the co-cultivation and transient transformation of young seedlings with Agrobacterium, was adapted and optimized in Catharanthus roseus. We investigated the optimal conditions for Gus expression by varying the Agrobacterium density (OD600 = 0.29 and 0.50), A. rhizogenes strain (15834 and R1000), and co-cultivation time in liquid (2, 12, or 24 h) followed by incubation time on solid media (1 or 2 days). Transformation efficiency was assessed quantitatively in terms of average GUS intensity per cotyledon surface area and percentage of cotyledons transformed. GUS staining was observed in 100% of cotyledons co-cultivated with A. rhizogenes (OD600 = 0.50) co-transformed with the Mas promoter-driven Gus and pSoup helper plasmids, in the presence of 0.01% v/v Silwet L-77 for 24 h in liquid followed by 2-days on solid media. In addition, we observed that co-cultivation with Agrobacterium strongly induced Zct1 and Orca3, two transcription factors known to regulate defense-related alkaloid biosynthesis in C. roseus. Homologous transcription factors regulate defense responses in many plant species. Therefore, possible induction of defense-related genes by Agro-mediated transformation should be a consideration in experimental design.

Agrobacterium tumefaciens mediated genetic transformation of Ocimum gratissimum: A medicinally important crop

“…This might be due the fact that high concentration of surfactant may alter membrane permeability and causes cytotoxicity. The surfactant has successfully been used to enhance the efficiency of genetic transformation by many workers (Sood et al, 2014;Weaver et al, 2014).…”

GATA and Phytochrome Interacting Factor Transcription Factors Regulate Light-Induced Vindoline Biosynthesis in Catharanthus roseus

“…To determine the role of CrGATA1 in vindoline biosynthesis, we transiently overexpressed CrGATA1 in C. roseus seedlings using the fast agro-mediated seedling transformation (FAST) method (Weaver et al, 2014). Expression of CrGATA1 and the five lightresponsive vindoline pathway genes was measured in the aerial parts of young seedlings infiltrated either with the empty vector control pCAMBIA1300 (EV) or the overexpression vector pCAMBIA1300-CrGATA1 (CrGATA1-OX).…”

“…For transient overexpression in C. roseus seedlings, CrGATA1, CrPIF1, CrPIF3, or CrPIF4/5 was cloned into a modified pCAMBIA1300 vector containing CaMV35S promoter and rbcS terminator. C. roseus seedlings were transiently transformed with the plasmids using the FAST method with some modifications (Weaver et al, 2014). Briefly, Agrobacterium tumefaciens GV3101 harboring the plasmid was grown on Luria-Bertani (LB) plates containing 100 mg mL 21 kanamycin, 50 mg mL 21 gentamicin, and 30 mg mL 21 rifampicin.…”

Agrobacterium-Mediated Plant Transformation: Biology and Applications

“…FAST was demonstrated to work for Catharanthus roseus seedling transformation. However, it is worth noting that the FAST method strongly induces host defence genes Zct1 and Orca3; this should be taken into account in experimental design (Weaver et al, 2014). Optimization of A. tumefaciens culture conditions can also improve seedling transformation efficiency.…”