Gene Expression Analysis of Microtubers of Potato Solanum tuberosum L. Induced in Cytokinin Containing Medium and Osmotic Stress

Abstract

Potato microtuber productions through in vitro techniques are ideal propagules for producing high quality seed potatoes. Microtuber development is influenced by several factors, i.e., high content sucrose and cytokinins are among them. To understand a molecular mechanism of microtuberization using osmotic stress and cytokinin signaling will help us to elucidate this process. We demonstrate in this work a rapid and efficient protocol for microtuber development and gene expression analysis. Medium with high content of sucrose and gelrite supplemented with 2iP as cytokinin under darkness condition produced the higher quantity and quality of microtubers. Gene expression analysis of genes involved in the two-component signaling system (StHK1), cytokinin signaling, (StHK3, StHP4, StRR1) homeodomains (WUSCHEL, POTH1, BEL5), auxin signaling, ARF5, carbon metabolism (TPI, TIM), protein synthesis, NAC5 and a morphogenetic regulator of tuberization (POTH15) was performed by qPCR real time. Differential gene expression was observed during microtuber development. Gene regulation of two component and cytokinin signaling is taking place during this developmental process, yielding more microtubers. Further analysis of each component is required to elucidate it.

Keywords: Solanum tuberosum; cytokinin signaling; microtuberization; potato; two component system.

Figure 1

(A). Microtuber development of potato S. tuberosum cv. Alpha in medium with 8% sucrose, 6 g/L gelrite and activated charcoal after four weeks of incubation in darkness. (B). Shoot explants of potato in medium with low content of sucrose (1%), gelrite 3 g/L and activated charcoal after four weeks of incubation in darkness. Bar represents 1 cm.

Figure 2

Gene network derived from STRING database of potato S. tuberosum (0.500 confidence) analyzed using qPCR. Homologous sequences of A. thaliana are written in rows. Numbers in the bubble indicate the gene identifier of each gene derived from the potato genome. The figure represents a full network, the edges indicate both functional and physical protein associations.

Figure 3

Gene expression analysis by qPCR during microtuber induction in osmotic stress medium (MR8-G6-2iP) after 8, 15, 23 and 31 dayss of incubation in darkness. Control medium was non-osmotic medium (MR1-G3-2iP). Relative expression levels plotted based on Log2. Normalized with EF1 α (elongation factor 1 α) and SEC3 (exocyst complex component 3).

Figure 4

A model of microtuberization regulatory network in two conditions; osmotic stress (MR8-G6-2iP) and non-osmotic stress (MR1-G3-2iP).