Temporal and spatial expression analysis of shoot-regeneration regulatory genes during the adventitious shoot formation in hypocotyl and cotyledon explants of tomato (CV. Micro-Tom)

Abstract

Enhancing the competence for plant regeneration in tissue culture studies is an important issue not only for efficient genetic transformation of commercial crops but also for the reproducibility of scientific reports. In this study, we investigated optimization of several tissue culture conditions including plant growth regulators, types and ages of explants, culture densities, and plant position in order to improve the competence of adventitious shoot formation of the tomato (Solanum lycopersicum cv. Micro-Tom). In addition, we examined the differential expression of D-type cyclin (CYCD3-1) and several shoot regeneration regulatory genes from hypocotyl and cotyledon explants of tomato during shoot organogenesis. A treatment of 1 mg L-1 Zeatin and 0.1 mg L-1 Indole-3-acetic acid (IAA) in Murashige and Skoog (MS) medium containing 3% sucrose was optimal for adventitious shoot formation from hypocotyl and cotyledon explants. The younger explants exhibited more shoot formation regardless of explant types. Additionally, those closest to the shoot apical meristem produced more shoots compared to the other regions in the hypocotyl and the cotyledon explants. Gene expression of CYCD3-1, SHOOT MERISTEMLESS (STM), and cytokinin dependent WUSCHEL (WUS) was significantly higher in younger explants than in older ones. Furthermore, an increase in CYCD3-1, STM, and WUS expression was evident at the distal part of hypocotyls and the proximal part of cotyledons compared to other regions. These differential gene expression profiles exhibited good agreement with the results of shoot formation obtained from diverse explants of tomato. These results suggest that temporal and spatial gene expression of shoot regeneration regulatory genes plays an important role in enhancing the competence and the reproducibility of adventitious shoot formation from tomato explants.