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.

Highly efficient Agrobacterium-mediated transformation and plant regeneration system for genome engineering in tomato

“…High concentrations of cytokinins and low auxins stimulate shoot buds, proliferation, and enhancement of shoot production ( Gupta and Rashotte, 2012 , Phillips and Garda, 2019 ). The media supplemented with ZEA and IAA was the most efficient cytokinin and auxin combination for shoot bud initiation and proliferation and plant regeneration in several genotypes of tomato ( Senapati, 2016 , Gupta and Van Eck, 2016 , Saeed et al, 2019 , Lee et al, 2020 ).…”

“…It is necessary to develop a proficient plant regeneration and genetic transformation system in different tomato genotypes resilient to biotic and abiotic stresses and yield high through genome editing tools ( Gerszberg et al, 2015 , Gupta and Van Eck, 2016 , Reem and Van Eck, 2019 , Van Eck, 2020 ). Plant regeneration methods have been reported from various genotypes using cotyledon, cotyledonary node, hypocotyl, leaf, node, shoot tip, stem, pistil, and inflorescence as explants ( Koul et al, 2014 , Senapati, 2016 , Alatar et al, 2017 , Lee et al, 2020 ). The different types of experiments with varying concentrations of hormones are designed based on previous studies ( Senapati, 2016 ).…”

“…The PGR concentrations and combinations play a crucial role during the in vitro morphogenesis of tomato culture. Other parameters also affect the tomato plant regeneration frequency, such as the age of explants, type of medium, and components ( Senapati, 2016 , Alatar et al, 2017 , Lee et al, 2020 ).…”

New Insights Into Tissue Culture Plant-Regeneration Mechanisms

“…Regardless of the explant, initial cell division begins at a young part near the cambium and vascular bundles. Explants in the juvenile-development phase are more regenerative and possess higher totipotency than those of adult explants ( Lee et al, 2020 ). For example, a study investigating the frequency of embryonic callus induction among different ages of maize seedlings found a higher frequency of embryonic callus induction for seedlings that were between 2- and 6-cm long than for longer seedlings ( Long et al, 2020 ).…”

Integrating the Roles for Cytokinin and Auxin in De Novo Shoot Organogenesis: From Hormone Uptake to Signaling Outputs

“…For certain plant species, either direct and/or indirect shoot organogenesis can be carried out in one step, without the need for pre-incubation on CIM. The requirement for CIM is dependent on plant species and genotype, but the same sequence of molecular events that accompany the stages of DNSO is likely to take place in competent explants regardless of whether they are physically incubated on CIM or not [22,[28][29][30]. The morphogenic events that are part of DNSO occur within a unique, highly organized cellular and genetic framework, for which the presence of plant growth regulators in the regeneration media is merely a condition that enables certain developmental events to switch on.…”