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.
Highlights
As of yet, no comparative studies have been published on the relationships between the types of tomato explants and the expression profiles of major regulatory genes involved in shoot organogenesis
We found that the segments close to the shoot apical meristem were more prone to adventitious shoot formation than segments further from them
Since age and position are important factors involved in shoot formation from hypocotyl and cotyledon explants of tomato, we examined gene-expression profiles of CYCD3-1 and major shoot-regeneration regulatory genes to verify whether temporal and spatial expression changes are related to the acceleration of adventitious shoot formation
Summary
Along with plant growth regulators, several tissue culture parameters play a crucial role in controlling the adventitious shoot regeneration potential of explants, including plant age [11,12,13], explant position [13], explant orientation (abaxial or adaxial) [14,15], and explant density [16,17]. The explants of younger plants and the parts closer to the shoot apical meristem demonstrated higher shoot formation rates and higher expression levels of CYCD3-1, STM, and WUS during cultivation These results provide an efficient and reproducible method for culturing tomato explants and key information of temporal and spatial differences in the expression of shoot-regeneration regulatory genes during adventitious shoot formation
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